Products and compositions

Abstract

The present invention provides nucleic acid products capable of modulating, in particular, interfering or inhibiting AGT gene expression. These products may be oligomeric compounds comprising at least a first region of linked nucleosides having at least one first nucleobase sequence that is at least partially complementary to at least a portion of RNA transcribed from the AGT gene wherein the first nucleobase sequence is selected from the group consisting of SEQ ID NOS: 1 to 100.

Description

[0001] Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 407,353, filed on September 16, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The above XML document was created on September 11, 2023, named 4690_0081i_SL.xml, and is 23,336,830 bytes in size.

[0005] Field

[0006] The present invention provides nucleic acid products that can modulate, interfere with, or inhibit the expression of the angiotensinogen (AGT) gene. Specifically, methods, compounds, and compositions for reducing AGT (mRNA) and protein expression in animals are provided. Such methods, compounds, and compositions can be used to treat, prevent, or ameliorate AGT-related diseases or disorders, such as hypertension.

[0007] Background

[0008] The renin-angiotensin-aldosterone system (RAAS) plays an important role in blood pressure regulation. The RAAS cascade begins with the release of renin into the circulation, particularly into the plasma, by well-known mechanisms. Active renin in the plasma catalyzes the cleavage of angiotensinogen (AGT) produced by the liver to generate angiotensin I. Angiotensin I is converted to angiotensin II by the action of angiotensin-converting enzyme (ACE) expressed in the circulation and locally. Angiotensin II is a peptide hormone that causes vasoconstriction, thereby increasing blood pressure and potentially leading to hypertension and related diseases.

[0009] Dysregulation of angiotensin II, particularly its overproduction, can trigger hypertension and may lead to increased oxidative stress, promote inflammation, hypertrophy, and fibrosis of the heart, kidneys, and arteries, and ultimately may result in left ventricular fibrosis, arterial remodeling, and glomerulosclerosis.

[0010] Diseases

[0011] Hypertension is the most common controllable disease in developed countries, affecting 20%-50% of the adult population. Hypertension is a major risk factor for a variety of diseases, disorders, and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (such as aortic aneurysm), peripheral artery disease, heart damage (such as cardiac enlargement or hypertrophy), and other cardiovascular-related diseases, disorders, or conditions. In addition, hypertension has been shown to be an important risk factor for cardiovascular morbidity and mortality, accounting for 62% of all stroke cases and 49% of all heart disease cases. In 2017, the guidelines for the diagnosis, prevention, and treatment of hypertension were changed to set lower blood pressure targets to further reduce the risk of hypertension-related diseases and disorders (for example, see "Systematic Review of the 2017 American College of Cardiology (ACC) / American Heart Association (AHA) / American Academy of Physician Assistants (AAPA) / ABC / American College of Preventive Medicine (ACPM) / American Geriatrics Society (AGS) / American Pharmacists Association (APhA) / American Society of Hypertension (ASH) / American Society of Preventive Cardiology (ASPC) / National Medical Association (NMA) / Physicians' Council for Responsible Medicine (PCNA) Guidelines for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines." Journal of the American College of Cardiology, November 7, 2017. Article number: S0735-1097(17)41517-8. doi:10.1016 / j.jacc.2017.11.004; and Whelton et al., ( "

[0012] "2017 ACC / AHA / AAPA / ABC / ACPM / AGS / APhA / ASH / ASPC / NMA / PCNA Guidelines for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines." Journal of the American College of Cardiology, November 7, 2017. Article number: S0735-1097(17)41519-1. doi:10.1016 / j.jacc.2017.11.006).

[0013] Hypertension may also occur together with diseases associated with proprotein convertase subtilisin / kexin type 9 (PCSK9) and / or apolipoprotein C3 (APOC3), such as dyslipidemia, and more specifically, hypercholesterolemia, hypertriglyceridemia, hyperchylomicronemia, and atherosclerotic cardiovascular disease (ASCVD).

[0014] Treatment

[0015] Although there are a variety of antihypertensive drugs available for the treatment of hypertension, more than two-thirds of patients cannot effectively control their blood pressure with only one antihypertensive drug and need to select two or more antihypertensive drugs from different drug classes. As the number of medications increases, patient compliance decreases and side effects increase, which further reduces the number of patients with controlled blood pressure. In addition, multiple studies have suggested a potential link between long-term use of antihypertensive drugs and worsening kidney function, and it has been found that the effect of antihypertensive drugs used to control blood pressure on kidney function is independent of their effect on blood pressure (Tomlinson et al. (2013), PLoS ONE 8(11), article number e78465; SPRINT Research Group (2015), The New England Journal of Medicine 373(22):2103-2116, ClinicalTrials.gov identifier NCT01206062; Kidney Disease: Improving Global Outcomes (KDIGO) Chronic Kidney Disease Work Group (2013), Kidney International Supplements 3:1-150; Kamarov et al., (2018, Hindawi Publishing Corporation, International Journal of Chronic Diseases, article number 1382705|https: / / doi.org / 10.1155 / 2018 / 1382705).

[0016] In addition, antihypertensive drugs, renal denervation, baroreceptor activation therapy, dietary changes, and lifestyle modifications may lower blood pressure and alleviate hypertension-related diseases, disorders, and / or conditions (Paulis et al., Nature Reviews Cardiology, 2012, 9:276-285). However, currently approved therapies for the treatment of hypertension have limitations because a significant portion of hypertensive patients are unable to achieve adequate blood pressure control. For example, drugs that target parts of the renin-angiotensin system (RAS) pathway, such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs), have limitations in their ability to inhibit the RAAS pathway (Nobakht et al., Nature Reviews Nephrology, 2011, 7:356-359). In addition, certain antihypertensive drugs, such as ACE inhibitors, are contraindicated in hypertensive patients with kidney disease because they have the potential to damage the patient's kidney function.

[0017] Accordingly, there is a need for additional compounds and methods of treatment that can effectively mitigate the dysregulatory effects (such as hypertension) caused by the overproduction of angiotensin II and overcome the above disadvantages.

[0018] Summary

[0019] The present invention provides compounds and methods of treatment that can effectively mitigate the dysregulatory effects (such as hypertension) caused by the overproduction of angiotensin II.

[0020] According to a first aspect, there is provided an oligomeric compound that inhibits the expression of angiotensinogen (AGT), the compound comprising at least a first region composed of linked nucleosides, the region having at least one first nucleoside base sequence that is at least partially complementary to at least a portion of the RNA transcribed from the AGT gene, wherein the first nucleoside base sequence is selected from the sequences in Table 1a (SEQ ID NOs: 1 to 100) or a portion thereof, the length of the portion being advantageously at least 18 nucleosides.

[0021] A particularly advantageous embodiment is an optimized hairpin RNA (referred to as mxRNA); further details are provided in the embodiments and discussions below.

[0022] The present invention also provides double-stranded RNA (dsRNA). Unlike mxRNA, dsRNA lacks the loop connecting the antisense and sense strand portions and thus comprises two strands. These two strands are not covalently linked but form a base-pairing double-stranded region.

[0023] In a second aspect, a nucleic acid construct may comprise at least:

[0024] (a) A first nucleic acid portion that is at least partially complementary to at least a first portion of the RNA transcribed from the AGT gene;

[0025] (b) A second nucleic acid portion that is at least partially complementary to at least a second portion of the RNA transcribed from the AGT gene, the second portion being different from the first portion;

[0026] (c) A third nucleic acid portion that is at least partially complementary to the first nucleic acid portion in (a) and thus forms a first nucleic acid double-stranded region therewith;

[0027] (d) A fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion in (b) and thus forms a second nucleic acid double-stranded region therewith.

[0028] Advantageous and / or exemplary features of the constructs according to this second aspect are as follows:

[0029] 1) They comprise multiple (two or more) at least partially double-stranded reagents capable of triggering RNA interference, mainly linked into a single nanostructure through complementary (Watson-Crick) interactions;

[0030] 2) Optionally, other (e.g.) covalent bonds can be used to construct the construct and / or add various ligands (e.g., delivery / targeting moieties such as N-acetylgalactosamine (GalNAc) and / or other carbohydrates, cholesterol, peptides, or small molecules, optionally linked through a linker);

[0031] 3) The construct mainly contains chemically modified nucleotides (such as 2'-fluoro (2’F), 2'-methoxy (2’OMe), locked nucleic acid (LNA), peptide nucleic acid (PNA), methoxyethoxy (MOE), bridged nucleic acid (BNA), phosphorodiamidate morpholino oligomers (PMO), phosphorothioate, dithiophosphate, etc.), mainly (but not limited to) for improving resistance to nucleases;

[0032] 4) The construct contains "fragile" components (such as chemical linkers, unmodified nucleotides, etc.), which enables the construct to decompose when exposed to certain biological environments (such as extracellular and / or intracellular fluids); specific examples can be (but not limited to): a) nucleases cut the oligomer backbone at unmodified nucleotide sites; b) chemical linkage bonds break due to pH changes (such as in endosomes);

[0033] 5) When exposed to certain biological environments, it decomposes and releases active components (such as at least partially double-stranded reagents capable of triggering RNA interference) to regulate (upregulate or downregulate, preferably downregulate) the expression of target genes in cells / organisms;

[0034] 6) These constructs can be used to regulate, preferably downregulate or silence gene expression to study gene function or treat various diseases related to the target gene to be downregulated.

[0035] In a third aspect, the composition comprises an oligomeric compound according to the first aspect and / or a nucleic acid construct according to the second aspect, and a physiologically acceptable excipient.

[0036] In a fourth aspect, there is provided a pharmaceutical composition comprising an oligomeric compound according to the first aspect and / or a nucleic acid construct according to the second aspect.

[0037] The fifth aspect provides an oligomeric compound according to the first aspect and / or a nucleic acid construct according to the second aspect for use in human or veterinary medicine or therapy.

[0038] The sixth aspect provides an oligomeric compound according to the first aspect and / or a nucleic acid construct according to the second aspect for use in a method of treating a disease or disorder.

[0039] The seventh aspect provides a method of treating a disease or disorder, the method comprising administering to an individual in need of treatment an oligomeric compound according to the first aspect and / or a nucleic acid construct according to the second aspect.

[0040] In an eighth aspect, the oligomeric compound according to the first aspect or the second aspect can be used as a gene function analysis tool in research.

[0041] In a ninth aspect, an oligomeric compound according to the first or second aspect is used for manufacturing a medicament for treating a disease or disorder.

[0042] Effects achieved by the oligomeric compound

[0043] As shown in the examples disclosed herein, the oligomeric compounds described herein can significantly reduce the gene expression of AGT. For example, when using 5-donor primary human hepatocytes in vitro, compared with the negative control, the most effective compound surprisingly reduces the AGT messenger ribonucleic acid (mRNA) expression by about 91% or more in vitro, even up to 94.8%. In addition, as shown in the examples, compared with the negative control, these compounds are capable of reducing AGT expression by at least 50% in vitro. Since the AGT expression can be successfully reduced, these compounds effectively mitigate the effects of overexpression of angiotensin II caused by the dysregulation of the RAAS cascade. The significant reduction of AGT will reduce the production of angiotensin II, thereby enabling the treatment of hypertension.

[0044] Furthermore, surprisingly, compared with traditional short hairpin RNA (shRNA) molecules with longer lengths, using an oligomeric compound in the form of an mxRNA construct with a length shortened to, for example, 33 nucleotides to inhibit the gene expression of AGT can achieve the above effects. For example, this can make the synthesis of mxRNA molecules more advantageous in terms of cost and production efficiency because fewer units are required.

[0045] For certain mxRNA constructs targeting AGT gene expression, it was surprisingly found that by using a short sense strand (advantageously having a length of 14 nucleotides, shorter than the sense strand length in traditional shRNA molecules) within the mxRNA, the above effects can be achieved.

[0046] In addition, the AGT-targeting nucleic acid sequences disclosed herein can also be used in combination with apolipoprotein C3 (APOC3)-targeting nucleic acid sequences or proprotein convertase subtilisin / kexin type 9 (PCSK9)-targeting nucleic acid sequences as part of a multi-target RNA (muRNA), as described below. This design is reasonable because mxRNAs containing the AGT-targeting nucleic acid sequence in the antisense strand form have been demonstrated to be capable of inhibiting AGT gene expression by in vitro experiments herein (see, for example, Examples 1 and 2). On the other hand, mxRNA constructs containing the APOC3-targeting nucleic acid sequence or the PCSK9-targeting nucleic acid sequence in the antisense strand form have been demonstrated to be active in inhibiting APOC3 gene or PCSK9 gene expression in in vitro and in vivo experiments, respectively (see, for example, Examples 3 to 11 herein). Without wishing to be bound by a particular theory, it is believed that when the antisense strands targeting AGT, APOC3, or PCSK9 become part of the muRNA nucleic acid construct, they are also the active components in the corresponding gene knockdown process. Therefore, the muRNA constructs disclosed herein, including the AGT-targeting antisense strand and the APOC3-targeting antisense strand, or the AGT-targeting antisense strand and the PCSK9-targeting antisense strand, are active in knocking down their respective target genes. This has also been demonstrated by the in vivo and in vitro experiments disclosed herein, which show that muRNA molecules containing the APOC3-targeting antisense strand and the PCSK9-targeting antisense strand are capable of achieving the corresponding target gene knockdown (see, for example, Examples 12 and 13). Without wishing to be bound by a particular theory, assuming that the active components of muRNA and mxRNA molecules are the same if they contain the corresponding targeting antisense strands, the experiments of the present application also demonstrate that muRNA molecules using the strands disclosed herein to target AGT, APOC3, and PCSK9 are capable of simultaneously and effectively knocking down the AGT, APOC3, and PCSK9 genes. Therefore, muRNA molecules can be used to treat diseases related to PCSK9 and / or APOC3, such as dyslipidemia, more specifically hypercholesterolemia, hypertriglyceridemia, hyperchylomicronemia, and atherosclerotic cardiovascular disease (ASCVD), while treating hypertension.

[0047] In addition, it has been found that when the muRNA construct contains the AGT-targeting nucleic acid portion and the APOC3-targeting portion disclosed herein, it can be used to simultaneously inhibit AGT and APOC3.

[0048] The effects and technical advantages achieved by using these oligomeric compounds to inhibit AGT expression will be presented in more detail in the detailed description and examples.

[0049] Brief Description of the Drawings

[0050] Figure 1Shows the single-dose curves of certain AGT mxRNA compounds and their activity in inhibiting AGT gene expression (primary screening), and these compounds are selected from Table 3c, with serial numbers 501 - 600 and 3604 - 3605.

[0051] Figures 2a to 2h Shows the dose curves of 30 AGT mxRNA compounds and their activity in inhibiting AGT gene expression (secondary screening). See Example 2.

[0052] Figure 2a : AGT_27, AGT_44, AGT_41, AGT_97 (serial numbers: 527, 544, 541, and 597);

[0053] Figure 2b : AGT_90, AGT_62, AGT_52, AGT_93 (serial numbers: 590, 562, 552, and 593);

[0054] Figure 2c : AGT_49, AGT_73, AGT_18, AGT_37 (serial numbers: 549, 573, 518, and 537);

[0055] Figure 2d : AGT_56, AGT_100, AGT_40, AGT_75 (serial numbers: 556, 5100, 540, and 575);

[0056] Figure 2e : AGT_30, AGT_42, AGT_81, AGT_17 (serial numbers: 530, 542, 581, and 517);

[0057] Figure 2f : AGT_34, AGT_53, AGT_29, AGT_26 (serial numbers: 534, 553, 529, and 526);

[0058] Figure 2g : AGT_74, AGT_94, AGT_14, AGT_3 (serial numbers: 574, 594, 514, and 503);

[0059] Figure 2h : AGT_7, AGT_2, TMPRSS, M4K4 (serial numbers: 507 and 502; the latter two are controls).

[0060] Figure 3a Shows the dose curves of apolipoprotein C3 (APOC3) lead compounds in primary human hepatocytes at different doses;

[0061] Figure 3bShows the dose curve of the APOC3 lead compound for humanized mouse studies in primary human hepatocytes as described in Examples 3 - 5.

[0062] Figure 4 Shows a schematic diagram of a study, including the time points of drug administration to mice and the time points of sampling, as described in Examples 6 and 7.

[0063] Figure 5 Shows the average percentage of remaining APOC3 messenger ribonucleic acid (mRNA) in liver tissue and the APOC3 protein level in plasma in animals treated with APOC3 - targeted mxRNA constructs [A28(14 - 4)mF mF (SEQ ID NOs: 2176 and 2188) and A277(12 - 5)mF (SEQ ID NOs: 2169 and 2181)] compared to the control group, as further described in Example 6.

[0064] Figure 6 Shows the average percentage of triglycerides (TG) and total cholesterol (TC) in serum in animals treated with APOC3 - targeted mxRNA constructs [A28(14 - 4)mF mF (SEQ ID NOs: 2176 and 2188) and A277(12 - 5)mF (SEQ ID NOs: 2169 and 2181)] compared to the control group, as further described in Example 6.

[0065] Figure 7a and Figure 7b Shows the average percentage of remaining APOC3 mRNA in liver tissue ( Figure 7a ) and the APOC3 protein level in plasma detected by enzyme - linked immunosorbent assay (ELISA) ( Figure 7b ) in animals treated with APOC3 - targeted mxRNA constructs [A28(14 - 4)mF mF (SEQ ID NOs: 2176 and 2188) and A277(12 - 5)mF (SEQ ID NOs: 2169 and 2181)] at weeks 2 and 6 compared to the control group, as further described in Example 6.

[0066] Figure 8a and Figure 8b Shows the average percentage of triglycerides ( Figure 8a ) and total cholesterol ( Figure 8b ) in serum in animals treated with APOC3 - targeted mxRNA constructs [A28(1 - 4)mF mF (SEQ ID NOs: 2176 and 2188) and A277(12 - 5)mF (SEQ ID NOs: 2169 and 2181)] at weeks 2 and 6 compared to the control group, as further described in Example 6.

[0067] Figure 9Shows a schematic diagram of an extended study of compound A28(14-4)mF(also known as STP125G)(sequence numbers: 2176 and 2188) using humanized liver mice(see also Figure 4 and Example 6), as described in Example 7. The humanized liver mouse model(n = 4) was tested by subcutaneous injection of 10 mg / kg of STP125G; the terminal time points were weeks 2, 4, 6, 8, and 10. Quantitative polymerase chain reaction(qPCR, detecting the mRNA of human angiotensinogen AGT), plasma ELISA(detecting human AGT protein), and triglyceride concentration determination were performed.

[0068] Figure 10 a and Figure 10 b show the knockdown of APOC3 mRNA in the liver and protein in the plasma at week 12, as described in Example 7.

[0069] Figure 11 a and Figure 11 b show the serum triglyceride(TG) and total cholesterol(TC) at week 8, as described in Example 7.

[0070] Figure 12 Is a schematic diagram showing the development of the humanized liver of the mice used in the study described in Example 7. Derived from the article by M. Grompe and S. Strom(2013) published in the journal Gastroenterology, Volume 145: 1209-1214.

[0071] Figure 13 Shows a table of the knockdown values of proprotein convertase subtilisin / kexin type 9(PCSK9) in hepatocellular carcinoma cell lines compared with the negative control. The nucleobase sequences and backbone(sugar, phosphate) modifications were selected from Table 7a(sequence numbers: 2189-2215, unligated ligand) and Table 7b(ligated ligand, sequence numbers: 2216-2242). An asymmetric 15-19 structure with an alternating chemical modification pattern was used.

[0072] Cell seeding: 10,000 HepG2 cells were seeded per well; transfection was mediated by Lipofectamine RNAiMax; transfection: The cells were incubated for 72 hours in Eagle's minimum essential medium(EMEM) containing 10% fetal bovine serum(FBS) without antibiotics using a compound at a concentration of 20 nM; gene expression was measured by quantitative polymerase chain reaction(qPCR, Taqman chemistry), adjusted according to the standard curve, and normalized to the reference gene GAPDH; the data were expressed as a percentage of gene expression in untreated cells(NT).

[0073] Figure 14Shows the half-maximal inhibitory concentration (IC 50 ) for PCSK9 knockdown using various PCSK9 constructs. The nucleobase sequences and backbone (sugar, phosphate) modifications are selected from Tables 7a and 7b, including cases with and without attached ligands (SEQ ID NOs: 2189-2242, and the sequences identified in Tables 8a-11a, as shown in Table 19). See Examples 8-9.

[0074] Figure 15 Shows the concentration-dependence of PCSK9 knockdown by a particularly advantageous double-stranded construct carrying an N-acetylgalactosamine (GalNAc) ligand (antisense strand: 19 nucleotides; sense strand: 14 nucleotides) [constructs P29 (SEQ ID NOs: 3588-3589), P57 (SEQ ID NOs: 3590-3591), P53 (SEQ ID NOs: 3592-3593), P44 (SEQ ID NOs: 3594-3595)], and control groups: transmembrane serine protease 6 (TMPRSS6) and mitogen-activated protein kinase kinase kinase kinase 4 (M4K4) G8, all at different concentrations. The nucleobase sequence of the 14-nucleotide sense strand is different from that given in the Sequence Listing (15 nucleotides), with the 5'-terminal nucleotide removed. The backbone (sugar, phosphate) modifications are shown in Table 20 and are optimized double-stranded constructs as described in Example 10. M4K4 and TMPRSS6 are used as negative controls (unrelated targets).

[0075] Figure 16 Shows a comparison of two double-stranded constructs as described in Example 10. Antisense strand: 19 nucleotides; sense strand: 15 nucleotides when labeled "(15)" (nucleobase sequence as shown in the Sequence Listing), otherwise 14 nucleotides [constructs P29 (SEQ ID NOs: 3588-3589), P52 (SEQ ID NOs: 2194, 2221), P29(15) (SEQ ID NOs: 3596-3597), P52(15)], and control groups: M4K4 and PC1a. The backbone (sugar, phosphate) modifications are shown in Table 20 (SEQ ID NOs: 3588-3597). PC1a: positive control. A molecule similar to inclisiran was used, differing only in that its ligand is trivalent GalNAc. This is to be consistent with the constructs carrying trivalent GalNAc ligands. M4K4 is used as a negative control.

[0076] Figure 17 Shows the concentration-dependence of PCSK9 knockdown by selected hairpin molecules (mxRNA). "14-5-14" indicates a 19-nucleotide antisense region linked to a 4-nucleotide sense region (linked to Figure 15...(one nucleotide shorter), where the 5 nucleotides at the 3'-end of the antisense region form the loop of the hairpin, and the molecule contains a double-stranded region of 14 base pairs formed by base pairing of 14 nucleotides at the 5'-end of the antisense region with the nucleobases of the sense region. The nucleobase sequence corresponding to the entire hairpin molecule is listed in Table _____ and includes experimental labels. The backbone (sugar, phosphate) modification is shown in Table 21 (sequence numbers: 3598 - 3601). TMPRSS6 was used as a negative control. See Example 11.

[0077] Figure 18 Figure 1 shows a schematic diagram of a study conducted in humanized liver mice to screen for candidate molecules. The humanized liver mouse model (n = 4 or 5 per group) was used to test by subcutaneous injection of 5, 10, and 30 mg / kg of AGT27AmxRNA (sequence number: 3604) and AGT - 52A mxRNA (sequence number: 3605); the terminal time point was 2 weeks. qPCR (detecting human AGT mRNA) and plasma ELISA (detecting human AGT protein) were performed. See Example 14.

[0078] Figures 19a and 19b show the liver knockdown (KD) (Figure 19a) and plasma protein (Figure 19b) data obtained two weeks after the study (designed as Figure 18 shown).

[0079] Figure 20 Figure 2 shows a schematic diagram of a study conducted in humanized liver mice. The study aimed to determine the duration of response of GalNAc - conjugated mxRNAs (AGT27A mxRNA, sequence number 3604; AGT - 52A mxRNA, sequence number 3605) targeting human angiotensinogen (AGT) messenger ribonucleic acid (mRNA) in the disclosed embodiments, as described in Example 15 in detail. The humanized liver mouse model (n = 5) was used and tested by subcutaneous injection of 30 mg / kg of AGT27A. The terminal time points of the experiment were set at weeks 2, 4, 6, 8, and 12. Quantitative polymerase chain reaction (qPCR, detecting human AGT mRNA) and plasma enzyme - linked immunosorbent assay (ELISA, detecting human AGT protein) were carried out during this period.

[0080] Figures 21a and 21b show the time - dependent data (duration of response) obtained in the study described in Example 15, where Figure 21a is liver - related data and Figure 21b is plasma protein - related data.

[0081] Detailed description and embodiments

[0082] The following describes more embodiments (entries) by way of example only. It should be understood that the benefits and advantages mentioned herein may apply only to a single embodiment or may apply to multiple embodiments. Embodiments are not limited to those that solve all or part of the problems described, or have all or part of the benefits and advantages described.

[0083] The features of different aspects and embodiments can be reasonably combined according to the actual situation, which is obvious to those skilled in the art, and these features can be combined with any aspect described below.

[0084] Definition

[0085] The following definitions apply throughout the text. In many cases, these definitions not only give the meaning of the term itself, but also list possible implementations, but these listings are not exhaustive.

[0086] Unless otherwise specifically defined, the terms, procedures, and techniques used in the fields of analytical chemistry, organic synthetic chemistry, medicinal chemistry, and pharmaceutical chemistry involved herein are well-known and commonly used in the art. Chemical synthesis and chemical analysis can employ standard techniques. Relevant techniques and procedures can be referred to the following documents: "Carbohydrate Modifications in Antisense Research" (edited by Sangvi and Cook, American Chemical Society, Washington, D.C., 1994); "Remington: The Science and Practice of Pharmacy" (Mack Publishing Company, Easton, Pennsylvania, 21st Edition, 2005); "Antisense Drug Technology: Principles, Strategies, and Applications" (edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida); and "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (2nd Edition, Cold Spring Harbor Laboratory Press, 1989). For any purpose, the above documents are incorporated herein by reference in their entirety. Where permitted, all patents, applications, published applications, other publications, and related data mentioned in this disclosure are also incorporated herein by reference in their entirety.

[0087] Unless otherwise specified, the following terms have the following meanings:

[0088] Excipient: As used herein, "excipient" refers to any compound or mixture of compounds added to the compositions provided herein and suitable for delivering oligomeric compounds.

[0089] Nucleoside: As used herein, "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (such as those found in DNA and RNA) and modified nucleosides. A nucleoside can be linked to a phosphate moiety, and a nucleoside linked to a phosphate by a phospho-linkage is also referred to as a "nucleotide". The structural features and / or lengths of the oligomeric compounds or nucleic acid constructs disclosed herein are expressed in terms of "nucleosides" or "nucleotides".

[0090] As used herein, "chemical modification" or "chemically modified" refers to a chemical difference in a compound compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. For oligonucleotides, chemical modification does not include the case where only the nucleobase sequence is different.

[0091] As used herein, "furanosyl" refers to a five-membered ring structure composed of four carbon atoms and one oxygen atom.

[0092] As used herein, "naturally occurring sugar moiety" refers to the ribofuranosyl contained in naturally occurring RNA or the deoxyribofuranosyl contained in naturally occurring DNA. The "naturally occurring sugar moiety" referred to herein is also referred to as the "unmodified sugar moiety". In particular, such "naturally occurring sugar moiety" or "unmodified sugar moiety" referred to herein has -H (for DNA sugar moiety) or -OH (for RNA sugar moiety) at the 2'-position of the sugar moiety, especially -H at the 2'-position of the sugar moiety (for DNA sugar moiety).

[0093] As used herein, "sugar moiety" refers to the naturally occurring sugar moiety of a nucleoside or a modified sugar moiety. As used herein, "modified sugar moiety" refers to a substituted sugar moiety or a sugar substitute.

[0094] As used herein, "substituted sugar moiety" refers to a furanosyl that has been substituted. Substituted sugar moieties include, but are not limited to, furanosyls having substituents at the 2'-position, 3'-position, 5'-position, and / or 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.

[0095] As used herein, "2'-substituted sugar moiety" refers to a furanosyl having a substituent other than H or OH at the 2'-position. Unless otherwise specified, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of a 2'-substituted sugar moiety does not form a bridge bond with another atom of the furanosyl ring).

[0096] As used herein, "MOE" refers to -OCH2CH2OCH3.

[0097] As used herein, "2'-F nucleoside" refers to a nucleoside having a fluorine atom at the 2'-position of the sugar. Unless otherwise specified, the fluorine atom in the 2'-F nucleoside is in the ribose configuration (substituting the OH in natural ribose). A fully 2'-fluorinated (ribose) oligonucleotide duplex that hybridizes to an RNA strand is not a substrate for ribonuclease H, while its analogs retain ribonuclease H activity.

[0098] As used herein, "sugar substitute" refers to a structure that does not contain a furanosyl group but can replace the naturally occurring sugar moiety in a nucleoside such that the resulting nucleoside subunits can be linked to each other and / or to other nucleosides to form an oligomeric compound capable of hybridizing to a complementary oligomeric compound. Such structures include rings having a different number of atoms than the furanosyl group (e.g., four-membered, six-membered, or seven-membered rings); replacing the oxygen atom in the furanosyl group with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both changing the number of atoms and replacing the oxygen atom. Such structures may also have a substitution pattern similar to that of the substituted sugar moiety (e.g., a bicyclic sugar substitute of a six-membered carbon ring optionally bearing additional substituents). Sugar substitutes also include more complex forms of sugar replacement (e.g., the acyclic system of peptide nucleic acid). Sugar substitutes include, but are not limited to, morpholino, cyclohexenyl, and cyclohexanol moieties.

[0099] As used herein, "bicyclic sugar moiety" refers to a modified sugar moiety having a 4- to 7-membered ring (including, but not limited to, a furanosyl group) with a bridge connecting two atoms of the 4- to 7-membered ring to form a second ring, thereby forming a bicyclic structure. In certain embodiments, this 4- to 7-membered ring is a sugar ring; in certain cases, the 4- to 7-membered ring is a furanosyl group; in certain such embodiments, the bridge connects the 2'-carbon atom and the 4'-carbon atom of the furanosyl group.

[0100] Nucleotide: As used herein, "nucleotide" refers to a nucleoside further bearing a phosphate linking group. Linked nucleosides: "Linked nucleosides" may or may not be linked by a phosphate bond and thus include, but are not limited to, "linked nucleotides". As used herein, "linked nucleosides" refers to nucleosides linked in a continuous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0101] As used herein, "nucleobase" refers to a group of atoms capable of attaching to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide and that can form a bond, more specifically a hydrogen bond, with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases can be naturally occurring or modified.

[0102] Unmodified nucleobase or naturally occurring nucleobase: Refers to the heterocyclic nucleobases naturally occurring in RNA or DNA, namely the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

[0103] As used herein, "modified nucleoside" refers to a nucleoside that has at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. The modified nucleoside can contain a modified sugar moiety and / or a modified nucleobase.

[0104] "Bicyclic nucleoside" or "BNA" refers to a nucleoside having a bicyclic sugar moiety. "Locked nucleic acid nucleoside" or "LNA" refers to a nucleoside having a bicyclic sugar moiety with a 4'-CH2-O-2' bridge.

[0105] "2'-substituted nucleoside" refers to a nucleoside having a substituent other than H or OH at the 2'-position of the sugar moiety. Unless otherwise specified, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0106] "Deoxynucleoside" refers to a nucleoside having a 2'-H furanosyl sugar moiety, as in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2'-deoxynucleoside may contain a modified nucleobase or may contain an RNA nucleobase (e.g., uracil).

[0107] As used herein, "oligonucleotide" refers to a compound having multiple linked nucleosides. In certain embodiments, the oligonucleotide contains one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0108] As used herein, "modified oligonucleotide" refers to an oligonucleotide that contains at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0109] Compared to a naturally occurring phosphodiester bond, a favorable modified internucleoside linkage can confer higher stability. "Stability" particularly refers to stability against hydrolysis, including resistance to enzyme-catalyzed hydrolysis by exonucleases and endonucleases.

[0110] For single-stranded oligomeric compounds, favorable positions for such modified internucleoside linkages include termini and hairpin loops. For example, when counting from the 5'-terminus, the internucleoside linkages connecting the first and second nucleosides, the second and third nucleosides, and / or when counting from the 3'-terminus, the internucleoside linkages connecting the first and second nucleosides, the second and third nucleosides are modified. Additionally, the linkage connecting the nucleoside at the 3'-terminus to a ligand (such as N-acetylgalactosamine (GalNAc)) can also be modified.

[0111] As described above, the hairpin loop of a single-stranded oligomeric compound is also a favorable position. Specifically, all, all but one, or most of the internucleoside linkages in the hairpin loop are modified. As used herein, "internucleoside linkages in the hairpin loop" refers to the linkages between nucleosides that do not participate in base pairing. For example, in a hairpin loop composed of five nucleosides, there are four internucleoside linkages between nucleosides that do not participate in base pairing. Advantageously, the term "internucleoside linkages in the hairpin loop" also encompasses the linkages connecting the stem and the loop, i.e., the linkages connecting nucleosides that participate in base pairing and nucleosides that do not participate in base pairing. Generally, there are two such positions in the hairpin structure and mxRNA.

[0112] Most preferably, the modified internucleoside linkages are present in both termini and the hairpin loop simultaneously.

[0113] As used herein, "linkage" or "linking group" refers to a group of atoms that connects two or more other groups of atoms.

[0114] "Internucleoside linkage" refers to the covalent linkage between adjacent nucleosides in an oligonucleotide.

[0115] As used herein, "naturally occurring internucleoside linkage" refers to the 3'-to-5' phosphodiester linkage.

[0116] As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than the naturally occurring internucleoside linkage. Specifically, the "modified internucleoside linkage" as referred to herein may include modified phosphorus-containing linking groups, such as phosphorothioate or dithiophosphate internucleoside linkages.

[0117] As used herein, "terminal internucleoside linkage" refers to the linkage between the last two nucleosides of an oligonucleotide or a defined region thereof.

[0118] As used herein, "phosphorus-containing linking group" refers to a linking group containing a phosphorus atom, which may include phosphorus-containing linking groups naturally present in RNA or DNA, such as phosphodiester linking groups, or modified phosphorus-containing linking groups not normally present in natural RNA or DNA, such as phosphorothioate or dithiophosphate linking groups. Thus, phosphorus-containing linking groups include, but are not limited to, phosphodiester, phosphorothioate, dithiophosphate, phosphonate, methylphosphonate, phosphoramidate, etc.

[0119] As used herein, "internucleoside phosphorus-containing linking group" refers to a phosphorus-containing linking group that directly connects two nucleosides.

[0120] "Oligomeric compound" refers to a polymeric structure having two or more substructures. In certain embodiments, the oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide. In certain embodiments, the oligomeric compound further comprises one or more conjugate groups, terminal groups, and / or ligands. In certain embodiments, the oligomeric compound consists only of oligonucleotides. In certain embodiments, the oligomeric compound comprises a backbone composed of one or more linked monomeric sugar moieties, wherein each linked monomeric sugar moiety is directly or indirectly linked to a heterocyclic base moiety. In certain embodiments, the oligomeric compound may also include monomeric sugar moieties not linked to a heterocyclic base moiety, thereby forming abasic sites. The oligomeric compound can be defined solely based on the nucleobase sequence, i.e., by specifying the sequence of A, G, C, U (or T). In this case, the structure of the sugar-phosphate backbone is not particularly limited and may or may not contain modified sugars and / or modified phosphates. On the other hand, the oligomeric compound can also be more comprehensively defined, i.e., by specifying not only the nucleobase sequence but also the structure of the backbone, particularly the modification status of the sugar (unmodified, 2'-OMe modified, 2'-F modified, etc.) and / or phosphate. mxRNA is a non-limiting example of an oligomeric compound.

[0121] As used herein, "nucleic acid construct" or "construct" refers to a combination of two or more (such as four) oligomeric compounds. These oligomeric compounds can be linked to each other by covalent bonds (such as the phosphodiester bonds present in natural nucleic acids or modified forms thereof disclosed herein) or by non-covalent bonds (such as hydrogen bonds, advantageously hydrogen bonds between nucleobases, such as Watson-Crick base pairing). In certain embodiments, it is advantageous for the construct to comprise four oligomeric compounds, two of which are linked by a covalent bond, thereby forming two nucleic acid strands that bind to each other by hydrogen bonds. The strands may be fully complementary, but this is not necessarily the case. Specifically, in an exemplary embodiment, an antisense strand targeting the first region of angiotensinogen (AGT) messenger ribonucleic acid (mRNA) is covalently linked to a sense strand of another double-stranded RNA molecule targeting AGT, and the antisense strand of the double-stranded RNA molecule targeting AGT mRNA is covalently linked to a sense strand of another double-stranded RNA molecule targeting AGT mRNA. Since the antisense and sense strands of the parental single-target RNA molecule do not need to be of the same length, and advantageously the antisense strand portion is longer than the sense strand portion, a construct comprises a central region in which the 3' regions of the antisense strand portions of the parental single-target RNA molecules face each other. In this region, base pairing usually does not occur or only occurs partially, but complete complementarity is not excluded. In addition, when the antisense and sense strand portions of the respective parental RNA molecules face each other, there is complementarity, advantageously complete complementarity or 1 to 2 mismatches. Multitarget RNA (muRNA) is a non-limiting example of a nucleic acid construct.

[0122] The term "chain" has its established meaning in the art and refers to a plurality of linked nucleosides, with no particular limitation on the mode of linkage, but including phosphodiester bonds and variant forms thereof disclosed herein. A chain can also be regarded as a plurality of linked nucleotides, in which case the linking bond is a covalent bond.

[0123] As used herein, "terminal group" refers to one or more atoms attached to the 3'-end or 5'-end (also referred to as the "terminus") of an oligonucleotide, or both ends. In certain embodiments, the terminal group comprises one or more terminal group nucleosides, and a "terminal nucleoside" refers only to a single nucleotide at the corresponding terminus (5'-end or 3'-end).

[0124] "Conjugate" or "conjugating group" refers to an atom or group of atoms attached to an oligonucleotide or oligomeric compound. In certain embodiments, the conjugating group attaches a ligand to a modified oligonucleotide or oligomeric compound. Generally, the conjugating group can alter one or more properties of the compound to which it is attached, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance characteristics.

[0125] In the context of a conjugating group, "conjugating linker" or "linker" refers to the portion of the conjugating group that contains any atom or group of atoms that covalently links the oligonucleotide to another portion of the conjugating group. In certain embodiments, the point of attachment to the oligomeric compound is the 3'-oxygen atom of the 3'-hydroxy group of the 3'-terminal nucleoside of the oligonucleotide. In certain embodiments, the point of attachment is the 5'-oxygen atom of the 5'-hydroxy group of the 5'-terminal nucleoside of the oligonucleotide. In certain embodiments, the bond used to form the linkage to the oligomeric compound is a cleavable bond. In certain such embodiments, such a cleavable bond constitutes all or part of the cleavable moiety.

[0126] In certain embodiments, the conjugating group comprises a cleavable moiety (e.g., a cleavable bond or a cleavable nucleoside) and a ligand moiety, which can comprise one or more ligands, such as a carbohydrate cluster moiety, such as an N-acetylgalactosamine (also referred to as "GalNAc") cluster moiety. In certain embodiments, the carbohydrate cluster moiety is defined by the number and type of ligands. For example, in certain embodiments, the carbohydrate cluster moiety comprises 2 GalNAc groups. For example, in certain embodiments, the carbohydrate cluster moiety comprises 3 GalNAc groups, which is a particularly advantageous case. In certain embodiments, the carbohydrate cluster moiety comprises 4 GalNAc groups. Such ligand moieties are attached to the oligomeric compound via a cleavable moiety such as a cleavable bond or a cleavable nucleoside. The ligands can be arranged in a linear or branched structure, such as a bi-antennary or tri-antennary structure. A preferred carbohydrate cluster has the following chemical formula:

[0127]

[0128] In this structural formula, one, two, or three phosphodiester bonds may also be replaced by phosphorothioate bonds.

[0129] As used herein, "cleavable moiety" refers to a bond or group that can be cleaved under physiological conditions. In certain embodiments, the cleavable moiety is cleaved intracellularly or in a subcellular compartment (such as an endosome or lysosome). In certain embodiments, the cleavable moiety is cleaved by an endogenous enzyme (such as a nuclease). In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable moiety is a phosphodiester bond.

[0130] As used herein, "cleavable bond" refers to any chemical bond that can be broken.

[0131] As used herein, "carbohydrate cluster" refers to a compound having one or more carbohydrate residues attached to a linking group.

[0132] As used herein, "modified carbohydrate" refers to any carbohydrate having one or more chemical modifications relative to a naturally occurring carbohydrate.

[0133] As used herein, "carbohydrate derivative" refers to any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0134] As used herein, "carbohydrate" refers to a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative. Carbohydrates are biomolecules that contain carbon (C), hydrogen (H), and oxygen (O) atoms. Carbohydrates can include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, such as one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties. A particularly advantageous carbohydrate is N-acetylgalactosamine.

[0135] As used herein, "chain" refers to an oligomeric compound having linked nucleosides.

[0136] As used herein, "single-stranded" or "single-strandedness" refers to an oligomeric compound having linked nucleosides that are linked in a continuous sequence without breaks therebetween. Such single strands may contain regions having sufficient self-complementarity to be able to form a stable hairpin structure self-double strand.

[0137] As used herein, "hairpin" refers to a single-stranded oligomeric compound that contains a double strand formed by base pairing between sequences that are in opposite directions and self-complementary within the strand.

[0138] As used herein, a "hairpin loop" refers to an unpaired loop formed by contiguous nucleotides in a hairpin, which is the result of self-complementary sequence hybridization. The structure formed looks like a loop or is U-shaped.

[0139] Specifically, short hairpin RNA, also denoted as shRNA, contains a double-stranded region and a loop that connects the regions forming the double strand. The end of the double-stranded region that does not carry the loop can be blunt-ended or carry 3' and / or 5' overhangs. Blunt-ended constructs are particularly advantageous. The term "shRNA" is more general than "mxRNA" defined below and may include compounds in which the loop is not formed by or not only formed by the antisense strand. Specifically, shRNA includes an antisense strand (also called the guide strand) complementary to a region of the target RNA, and a sense strand (i.e., the passenger strand) substantially complementary to the antisense strand. More specifically, the antisense and sense strands in shRNA are directly linked, for example, by a phosphate or phosphorothioate, or by a third part of the loop formed by contiguous nucleotides, meaning that the 3' end of the antisense strand is covalently linked to the 5' end of the sense strand through covalent bonds with several other groups. Such direct linkage does not include nicks or cuts.

[0140] As used herein, "directionality" refers to the chemical head-to-tail orientation of an oligonucleotide determined based on the chemical convention of numbering the carbon atoms of the sugar moiety, i.e., there is a 5' end defined by the 5' carbon of the sugar moiety, and a 3'

[0141] end defined by the 3' carbon of the sugar moiety. In a double-stranded or double-stranded oligonucleotide, the strands are arranged in opposite 5' to 3' directions so that base pairing can occur between them.

[0142] As used herein, "double-stranded", also abbreviated as "dup", refers to two or more complementary strand regions or strands of an oligonucleotide or oligonucleotides, hybridized together through their non-covalent, sequence-specific interactions. Most commonly, hybridization in a double strand occurs between the nucleobases adenine (A) and thymine (T), and / or adenine (A) and uracil (U), and / or guanine (G) and cytosine (C). A double strand can be part of a single-stranded structure, in which case self-complementarity results in hybridization; or it can be the result of hybridization between the strands in a double-stranded construct.

[0143] As used herein, "duplex" or "duplexed" refers to a pair of oligomeric compounds that hybridize to each other. In certain embodiments, the duplex oligomeric compounds comprise a first and a second oligomeric compound.

[0144] As used herein, "expression" refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of a 5' cap), and translation.

[0145] As used herein, "transcription" or "transcribed" refers to the first of several steps in DNA-based gene expression, in which the target sequence of DNA is enzymatically copied into RNA (specifically mRNA). Through this transcription process, RNA polymerase reads the target sequence of DNA and synthesizes a complementary and antiparallel RNA sequence, i.e., the primary transcript.

[0146] As used herein, "target sequence" refers to the sequence to which an oligomeric compound is expected to hybridize to have an intended effect on angiotensinogen (AGT) expression. The oligonucleotide has sufficient complementarity with its target sequence to ensure hybridization under physiological conditions.

[0147] As used herein, "nucleobase complementarity" or "complementarity" when referring to nucleobases means a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T); in RNA, adenine (A) is complementary to uracil (U); in both DNA and RNA, guanine (G) is complementary to cytosine (C). In certain embodiments, complementary nucleobases refer to the nucleobases in an oligomeric compound that can base pair with the nucleobases of the target sequence. For example, if the nucleobase at a certain position in an oligomeric compound can form a hydrogen bond with the nucleobase at a certain position in the target sequence, then the hydrogen bond binding position of the oligomeric compound and the target sequence at this nucleobase pair is considered complementary. Nucleobases with specific modifications may still retain the ability to pair with the corresponding nucleobases and thus still have nucleobase complementarity.

[0148] As used herein, "non-complementary" when referring to nucleobases means a pair of nucleobases that cannot form a hydrogen bond with each other.

[0149] As used herein, "complementary" when referring to an oligomeric compound (such as linked nucleosides, oligonucleotides) means the ability of such an oligomeric compound or its region to hybridize to the target sequence or a region of the oligomeric compound itself through nucleobase complementarity.

[0150] The complementary oligomeric compound does not have to have nucleobase complementarity at every nucleoside, and a certain degree of mismatch is tolerable. In certain embodiments, the complementary oligomeric compound or region is complementary at 70% of the nucleobases (i.e., 70% complementary); in certain embodiments, the degree of complementarity is 80%; in certain embodiments, 90%; in certain embodiments, at least 95%; in certain embodiments, up to 100% complementary.

[0151] As used herein, the term "self-complementarity" when referring to an oligomeric compound means the property of a compound to fold upon itself and form a duplex through hybridization of nucleobases in internal complementary strand regions. Depending on the distance and length between the strand regions, the compound may form hairpin loops, junctions, bulges, or internal loops.

[0152] As used herein, "mismatch" means that when an oligomeric compound is aligned with a target sequence and / or the self-complementary region of the oligomeric compound, the nucleobases in the oligomeric compound fail to pair with the nucleobases at the corresponding positions in the target sequence, or when the oligomeric compound hybridizes due to self-complementarity, they fail to pair with the nucleobases at the corresponding positions in itself.

[0153] As used herein, "hybridization" means the pairing between complementary oligomeric compounds (e.g., an oligomeric compound and its target sequence). Although not limited to a specific mechanism, the most common pairing mechanism is the formation of hydrogen bonds between complementary nucleobases, which can be Watson-Crick hydrogen bonds, hoogsteen hydrogen bonds, or reverse hoogsteen hydrogen bonds.

[0154] As used herein, "specific hybridization" means the ability of an oligomeric compound to hybridize to one nucleic acid site with a higher affinity than to another nucleic acid site.

[0155] As used herein, "fully complementary" when referring to an oligomeric compound or a region thereof means that each nucleobase of the oligomeric compound or the region thereof is capable of pairing with the nucleobases of a complementary nucleic acid target sequence or the self-complementary region of the oligomeric compound. Thus, a fully complementary oligomeric compound or a region thereof has no mismatched or unhybridized nucleobases relative to its target sequence or the self-complementary region of the oligomeric compound.

[0156] As used herein, "percent complementarity" means the percentage of nucleobases in an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. The percent complementarity is calculated by dividing the number of nucleobases in the oligomeric compound that are complementary to the nucleobases at the corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

[0157] As used herein, "percent identity" means the number of nucleobases in a first nucleic acid that have the same type (regardless of chemical modification) as the nucleobases at the corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0158] As used herein, "regulation" means a change in the quantity or quality of a molecule, function, or activity compared to before regulation. For example, regulation includes an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.

[0159] As used herein, the term "type of modification" when referring to a nucleoside or a "type" of nucleoside means a chemical modification of the nucleoside, including modified and unmodified nucleosides. Thus, unless otherwise indicated, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0160] As used herein, "different modifications" means chemical modifications or chemical substituents that are different from each other, including the case of no modification. Thus, for example, a methoxyethoxy (MOE)-modified nucleoside and an unmodified natural RNA nucleoside are "differently modified", even though the natural nucleoside is unmodified. Similarly, DNA and RNA oligonucleotides are "differently modified", even though they are both naturally occurring unmodified nucleosides. Nucleosides that are identical except for the nucleobase are not differently modified. For example, a nucleoside having a 2'-O-methyl (2'-OMe)-modified sugar moiety and an unmodified adenine nucleobase, and a nucleoside having a 2'-OMe-modified sugar moiety and an unmodified thymine nucleobase are not differently modified.

[0161] As used herein, "the same type of modification" means modifications that are the same as each other, including the case of no modification. Thus, for example, two unmodified RNA nucleosides have "the same type of modification", even though these RNA nucleosides are unmodified. Such nucleosides having the same type of modification can contain different nucleobases.

[0162] As used herein, "region" or "portion" means a plurality of contiguous nucleosides having a function or characteristic as defined herein, with particular reference to the claims and definitions provided herein. Generally, such a region or portion contains at least 10, at least 11, at least 12, or at least 13 contiguous nucleosides. For example, such a region can contain 13 to 20 contiguous nucleosides, such as 13 to 16 or 18 to 20 contiguous nucleosides. Generally, the first region as defined herein consists essentially of 18 to 20 nucleosides, and the second region as defined herein consists essentially of 13 to 16 contiguous nucleosides.

[0163] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0164] As used herein, "substituent" and "substituent group" refer to an atom or group of atoms that replaces an atom or group of atoms in a named parent compound. For example, a substituent that modifies a nucleoside is any atom or group of atoms that is different from the atoms or groups of atoms in a naturally occurring nucleoside (e.g., a modified 2'-substituent is any atom or group of atoms other than hydrogen or a hydroxyl group at the 2'-position of the nucleoside). Substituent groups can be protected or unprotected. In certain embodiments, the compounds of the present disclosure have substituents at one or more positions of the parent compound. Substituents can also be further substituted by other substituent groups and can be attached directly or through a linking group (such as oxygen or an alkyl or hydrocarbon group) to the parent compound.

[0165] Such substituents can be present as modifications on the sugar moiety, particularly substituents at the 2'-position of the sugar moiety. Unless otherwise indicated, groups suitable as substituents include, but are not limited to, one or more of the following: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl, carboxyl, alkoxy, alkoxyalkylidene, and amino substituents. Certain substituents described herein can represent a modification directly attached to the sugar moiety ring (such as a halogen directly attached to the sugar ring, such as fluorine), or a modification indirectly attached to the sugar moiety ring through an oxygen linking atom directly attached to the sugar moiety (such as an alkoxyalkylidene attached to an oxygen atom, such as methoxyvinyl, which generally provides a methoxyethoxy (MOE) substituent attached to the 2'-position of the sugar moiety as described herein).

[0166] As used herein, "alkyl" refers to a saturated straight-chain or branched-chain monovalent C1-6 hydrocarbon group, and methyl is a typical alkyl substituent at the 2'-position of the sugar moiety. The alkyl group is typically attached to the oxygen linking atom at the 2'-position of the sugar, and thus generally provides an -Oalkyl substituent on the sugar moiety of an oligomeric compound as described herein, such as an -OCH3 substituent.

[0167] As used herein, "alkylidene" refers to a saturated straight-chain or branched-chain divalent hydrocarbon group of the general formula -C n H 2n -, where n is 1-6. Methylene or ethylene is a typical alkylidene.

[0168] As used herein, "alkenyl" refers to a straight-chain or branched-chain unsaturated monovalent C2-6 hydrocarbon group, and vinyl or propenyl are the most typical alkenyl substituents at the 2'-position of the sugar moiety. As is well known to those skilled in the art, the degree of unsaturation in an alkenyl group is manifested by the presence of at least one carbon-carbon double bond. The alkenyl group is typically attached to the oxygen linking atom at the 2'-position of the sugar, and thus generally provides an -Oalkenyl substituent on the sugar moiety of an oligomeric compound as described herein, such as an -OCH2CH=CH2 substituent.

[0169] As used herein, "alkynyl" refers to a straight-chain or branched-chain unsaturated C2-6 hydrocarbon group, and ethynyl is a typical alkynyl substituent at the 2'-position of the sugar moiety. Those skilled in the art will appreciate that the degree of unsaturation in the alkynyl group is manifested by the presence of at least one carbon-carbon triple bond. The alkynyl group is usually attached to the oxygen-bonded atom at the 2'-position of the sugar, and thus, generally provides an -O-alkynyl substituent on the sugar moiety of the oligomeric compounds described herein.

[0170] As used herein, "carboxyl" refers to a group having the general formula -CO2H.

[0171] As used herein, "acyl" refers to a group formed by removing a hydroxyl group from the carboxyl group as defined herein, and has the general formula -C(O)-X, where X is usually a C1-6 alkyl group.

[0172] As used herein, "alkoxy" refers to a group formed by an alkyl group (such as a C1-6 alkyl group) and an oxygen atom, and this oxygen atom is used to attach the alkoxy group to the parent molecule (such as the 2'-position of the sugar moiety) or another group as defined herein (such as an alkylene group). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, and tert-butoxy. The alkoxy groups used herein may optionally contain other substituents.

[0173] As used herein, "alkoxyalkylene" refers to a group formed by attaching the alkoxy group as defined herein to the alkylene group as also defined herein, where the oxygen atom of the alkoxy group is attached to the alkylene group, and the alkylene group is then attached to the parent molecule. The alkylene group is usually attached to the oxygen-bonded atom at the 2'-position of the sugar, and thus, generally provides an -O-alkylenealkoxy substituent on the sugar moiety of the oligomeric compounds described herein, such as an -OCH2CH2OCH3 substituent. This is commonly referred to as the methoxyethoxy (MOE) substituent as defined herein and known in the art.

[0174] As used herein, "amino" includes primary amino, secondary amino, and tertiary amino.

[0175] As used herein, "halo" and "halogen" refer to atoms selected from fluorine, chlorine, bromine, and iodine.

[0176] As used herein, "mxRNA" is specifically understood according to the definition in WO 2020 / 044186 A2, the entire text of which is incorporated herein by reference. Specifically, mxRNA is a hairpin-shaped RNA molecule composed of an antisense portion (also referred to as the guide strand) and a sense portion (also referred to as the passenger strand). mxRNA contains a double-stranded region and a hairpin loop, and its length is approximately 34 nucleotides. The double-stranded region contains a region where a portion of the antisense portion base pairs with substantially the entire sense portion (usually 14 or 15 nucleotides per strand). The hairpin loop connects the two regions of the double strand (i.e., the antisense region and the sense region) through, for example, a phosphate or phosphorothioate linker (i.e., a covalent connection), and the antisense portion is usually about 18 to 20 nucleotides in length, thus forming an antisense double-stranded region and a loop. The loop where the antisense portion is located also connects the sense portion and the antisense portion of the second strand that forms the loop.

[0177] The term "angiotensinogen", abbreviated as "AGT", is also known as serine protease inhibitor family A member 8 (SERPINA 8) or human angiotensinogen (ANHU), and its meaning is consistent with the conventional understanding, referring to a protein produced by the liver. It is a component of the renin-angiotensin-aldosterone system (RAAS), and after being released into the bloodstream, it is converted into angiotensin I by renin. Angiotensinogen is expressed and produced by the angiotensinogen gene (i.e., the "AGT gene") in the liver.

[0178] As used herein, the term "muRNA" or "multi-RNA" refers to a nucleic acid construct that contains more than one, usually two, RNA sequences, namely the first and second nucleic acid portions, which target different regions of the AGT messenger ribonucleic acid (mRNA), or target the mRNA region of the AGT mRNA and the mRNA region of another target molecule. These targeting RNA sequences are also referred to as "antisense" strands or "guide" strands, and the respective passenger strands, namely the third and fourth nucleic acid portions that are complementary to the first and second portions respectively, are also included in the nucleic acid construct. Specifically, such muRNAs are designed such that after in vivo administration, they will break down and release the first and second nucleic acid portions. A specific example of such a muRNA is shown below, where (1) is the first nucleic acid portion, (2) is the third nucleic acid portion complementary to (1), (3) is the second nucleic acid portion complementary to the fourth nucleic acid portion, (5) is an unstable linker, and (6) is a ligand, and the latter two will be explained in detail below.

[0179]

[0180] It should also be understood that the oligomeric compounds described herein may have one or more non-hybridizing nucleosides (overhangs) at one or both ends of one or both strands, and / or one or more internal non-hybridizing nucleosides (mismatches), provided that there is sufficient complementarity to maintain hybridization under physiologically relevant conditions. Alternatively, at least one end of the oligomeric compounds described herein may be blunt-ended.

[0181] As used herein, "proprotein convertase subtilisin / kexin type 9 (PCSK9)" is a serine protease involved in lipid metabolism. PCSK9 reduces the number of low-density lipoprotein (LDL) receptors on the surface of hepatocytes. Therefore, an increase in the content and / or activity of PCSK9 leads to an increase in the level of "harmful" LDL cholesterol in the blood. Based on this molecular and cellular function of PCSK9, it has been recognized as a therapeutic target molecule.

[0182] As used herein, "APOC3" refers to apolipoprotein C3, which is secreted by the liver and small intestine. It is present in triglyceride-rich lipoproteins, including very low density lipoprotein (VLDL) and chylomicrons. It is involved in the negative regulation of lipid catabolism (especially triglyceride catabolism) and the clearance processes of VLDL, LDL, and high density lipoprotein (HDL). One molecular function of APOC3 is to inhibit lipoprotein lipase and hepatic lipase.

[0183] The use of the word "comprising" or "including" herein means including the identified method steps or elements, but these steps or elements are not an exhaustive list, so there may be additional steps or elements.

[0184] Short hairpin RNA (shRNA) and mxRNA oligomeric compounds

[0185] In a first aspect, there is provided an oligomeric compound capable of inhibiting the expression of angiotensinogen (AGT). The compound comprises at least a first contiguous nucleoside region having at least one first nucleoside base sequence that is at least partially complementary to a portion of the RNA transcribed from the AGT gene. The first nucleoside base sequence is selected from the sequences in Table 1a (SEQ ID NOs: 1 to 100) or a portion thereof, and the length of this portion is advantageously at least 18 nucleosides. Specifically, the 5'-terminal nucleoside of the first nucleoside base sequence may be replaced with uracil (U) instead of adenine (A), U instead of guanine (G), or U instead of cytosine (C).

[0186] In certain embodiments, the oligomeric compound may further comprise at least a second contiguous nucleoside region having at least one second nucleobase sequence that is at least partially complementary to the first nucleobase sequence and is selected from the sequences in Table 1b (SEQ ID NOs: 101 to 200) or a portion thereof, the length of the portion advantageously being at least 8, 9, 10, or 11, more advantageously at least 10 nucleosides. Specifically, the 3'-terminal nucleoside of the second nucleobase sequence may be replaced with adenine (A) for U, G, or C, respectively; more specifically, the nucleobase A serves as the nucleobase complementary to the 5'-terminal nucleoside of the first nucleobase sequence.

[0187] The first contiguous nucleoside region is also referred to as the antisense region or guide region / strand, and the second contiguous nucleoside region is referred to as the sense region or passenger region / strand. As disclosed in certain embodiments below, these two regions may be located on the same RNA strand, advantageously arranged adjacent to each other, thus forming a hairpin molecule, also referred to as mxRNA. On the other hand, these two regions may be located on different strands, thus forming double-stranded RNA (dsRNA), advantageously each strand consisting only of the corresponding region.

[0188] Without wishing to be bound by theory, it is assumed that the above oligomeric compound comprising the first contiguous nucleoside region and the second contiguous nucleoside region will be incorporated into the RNA-induced silencing complex (RISC) during the RNA interference process. Subsequently, the RISC assembly binds and degrades the target mRNA. Specifically, this process is achieved when the guide strand pairs with the complementary sequence in the AGT mRNA molecule and is induced to cleave by the catalytic component Ago2 of RISC. Therefore, since the expression of AGT is inhibited, it is believed that the effects associated with the overregulation of angiotensin II in the above pathway will also be inhibited.

[0189] In certain embodiments, the first nucleobase sequence is selected from the sequences in SEQ ID NOs: 27, 44, 41, 97, 90, 62, 52, 93, 49, 73, 18, 37, 56, 100, 40, 75, 30, 42, 81, 17, 34, 53, 29, 26, 74, 94, 14, 3, 7, and 2 or a portion thereof. Advantageously, the first nucleobase sequence is selected from the sequences in SEQ ID NOs: 27, 52, 56, 62, 75, and 93, particularly SEQ ID NOs: 27 and 52.

[0190] The second nucleoside base sequence can, for example, be selected from the following sequences or a part thereof: SEQ ID NO: 127, 144, 141, 197, 190, 162, 152, 193, 149, 173, 118, 137, 156, 200, 140, 175, 130, 142, 181, 117, 134, 153, 129, 126, 174, 194, 114, 103, 107, and 102. Advantageously, the second nucleoside base sequence is selected from the following sequences or a part thereof: SEQ ID NO: 127, 152, 156, 162, 175, and 193.

[0191] Length and molecular characteristics of the oligomeric compound of the first aspect

[0192] The first linked nucleoside region can substantially consist of 18 to 35, advantageously 18 to 20, more advantageously 18 or 19, and even more advantageously 19 linked nucleosides. In addition, the second linked nucleoside region can substantially consist of 10 to 35, 10 to 20, 10 to 16, 10 to 15, or 13, 14, or 15 linked nucleosides.

[0193] The oligomeric compound comprising the first and second linked nucleoside regions can contain at least one complementary double-stranded region, which contains at least a part of the first linked nucleoside region that is directly or indirectly linked to at least a part of the second linked nucleoside region. Advantageously, the length of the double-stranded region is 10 to 19, 12 to 19, 12 to 15, or 14 or 15 base pairs, and an optional mismatch can be present within the double-stranded region.

[0194] In certain embodiments, the first and second linked nucleoside regions each have a 5' to 3' directionality, thereby determining their 5' and 3' regions, respectively.

[0195] In the oligomeric compound with 5' to 3' directionality of the first and second linked nucleoside regions, the 5' region of the first linked nucleoside region can be directly or indirectly linked to the 3' region of the second linked nucleoside region, for example, by complementary base pairing. Advantageously, the 5'-terminal nucleoside of the first nucleoside region base-pairs with the 3'-terminal nucleoside of the second nucleoside region.

[0196] In the above embodiments, the 3' region of the first linked nucleoside region can be directly or indirectly linked to the 5' region of the second linked nucleoside region. Advantageously, the first nucleoside region is directly covalently linked to the second nucleoside region through, such as, a phosphate, phosphorothioate, or dithiophosphate. More advantageously, the 3'-terminal nucleoside of the first linked nucleoside region is directly covalently linked to the 5'-terminal nucleoside of the second linked nucleoside region through a phosphate, phosphorothioate, or dithiophosphate. Particularly advantageously, the 3'-terminal nucleoside of the first region is directly linked to the 5'-terminal nucleoside of the second region through a phosphorothioate internucleoside linkage.

[0197] This is equivalent to forming a single-stranded oligonucleotide directly fused from two regions. Due to the base pairing defined in the previous embodiment, such oligonucleotides will exhibit a hairpin configuration. The hairpin structure optimized in terms of size and the like is the subject of the following further embodiments.

[0198] In certain embodiments, the oligomeric compound may consist of a first contiguous nucleoside region and a second contiguous nucleoside region.

[0199] Each region may constitute a separate strand, thereby forming double-stranded RNA (dsRNA). Particularly advantageous dsRNAs are those with a first strand length of 19 nucleosides and a second strand length of 14 or 15, preferably 14, nucleosides. When defining the length of a region or strand, the two terms "nucleoside" and "nucleotide" (sometimes abbreviated as "nt") are used interchangeably.

[0200] As described above, the two regions can also be fused together to form a hairpin structure.

[0201] In certain embodiments, there may be a third contiguous nucleoside region acting as a spacer between the first region and the second region.

[0202] The oligomeric compound may comprise or consist of a single strand having or consisting of a first, a third, and a second nucleoside region, wherein at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region, thereby forming at least a partially complementary double-stranded region.

[0203] In other words, the oligomeric compound comprises a single strand having a first and a second nucleoside region, with at least a portion of the first nucleoside region directly or indirectly linked to at least a portion of the second nucleoside region to form at least a partially complementary double-stranded region. As described above, the third region is optional.

[0204] In certain embodiments, the oligomeric compound may comprise or consist of a single strand having or consisting of a first and a second contiguous nucleoside region, wherein at least a portion of the first contiguous nucleoside region is directly or indirectly linked to at least a portion of the second contiguous nucleoside region, thereby forming at least a partially complementary double-stranded region.

[0205] In such an oligomeric compound that may comprise or consist of a single strand, the first and second nucleoside regions are directly adjacent on the single strand.

[0206] In certain embodiments, the number of contiguous nucleosides in the first nucleoside region may be more than that in the second nucleoside region.

[0207] Optionally, the ratio of the total number of linked nucleosides in the first nucleoside region to the total number of linked nucleosides in the second nucleoside region is from about 19 / 15 to about 19 / 8, or from about 18 / 15 to about 18 / 8. In particularly advantageous embodiments, the ratio is 19 / 15, 19 / 14, 19 / 13, 18 / 15, 18 / 14 or 18 / 13.

[0208] Alternatively, in addition, the percentage of the total number of linked nucleosides in the first nucleoside region relative to the total number of nucleosides of the oligomeric compound can be from about 55% to about 60%. The percentage can be from 57% to about 59.5%, most advantageously the percentage is about 57.6% or about 59.4%.

[0209] Without wishing to be bound by theory, it is assumed that the above ratios and / or percentages provide a suitable ratio / percentage for the RISC complex to process the number of nucleotides in the mxRNA strand, and thus contribute to effective knockdown of AGT.

[0210] In an oligomeric compound where the number of linked nucleotides in the first region is more than that in the second region, the additional linked nucleosides in the first nucleoside region form a hairpin loop connecting the first and second linked nucleoside regions. Advantageously, the part of the first nucleoside base sequence complementary to the RNA transcribed from the AGT gene forms a hairpin loop, and the loop contains 2 to 5, advantageously 4 or 5 nucleosides.

[0211] Such compounds are also referred to herein as hairpin structures or mxRNA. Since the second region is shorter than the first region, compared with traditional small interfering RNAs (siRNAs) having two regions of similar length, the compound is optimized (or miniaturized) in size.

[0212] Advantageously, the hairpin loop contains 4 or 5 linked nucleosides. A particularly advantageous structure is that the first region has a length of 19 nucleosides, the second region has a length of 14 nucleosides, and the hairpin loop has a length of 5 nucleosides, where the 5 nucleosides in the hairpin loop are the 5 3'-terminal nucleosides of the first region. This molecular architecture of the hairpin structure or mxRNA is also labeled as "14-5-14" herein.

[0213] In certain embodiments, the oligomeric single strand as described above can be selected from Table 2, particularly from the group consisting of SEQ ID NOs: 227, 252, 256, 262, 275, 293 and 3602-3603. Advantageously, the nucleoside base of the 5'-terminal nucleoside of the first linked nucleoside region is replaced with uracil (U), and the nucleoside base of the 5'-terminal nucleoside of the second linked nucleoside region is replaced with adenine (A).

[0214] In certain embodiments, the single strand is selected from Table 3c, particularly from the group consisting of construct numbers 527, 552, 556, 562, 575, 593, and 3604 - 3605. Advantageously, the nucleobase of the 5'-terminal nucleoside of the first contiguous nucleoside region is replaced by U, and the nucleobase of the 5'-terminal nucleoside of the second contiguous nucleoside region is replaced by A.

[0215] In certain embodiments, a hairpin loop as described above may be present at the 3'-end of the first contiguous nucleoside region. If the nucleobases of one, two, or more 3'-terminal nucleosides of the first nucleobase sequence permit, these nucleosides can fold back and form or participate in forming the second contiguous nucleoside region. This structural design is also referred to as "spillover". This can occur only when there is self-complementarity between the nucleobases at the 3'-end of the guide sequence region in the double strand and the most 3'-terminal nucleobase of the same guide sequence. For example, a "13 - 5 - 13" design can be employed to further achieve miniaturization. The first "13" refers to the guide sequence region participating in double strand formation, 5 is the length of the loop also formed by the guide sequence, and the second "13" refers to the second region of the double strand, consisting of one nucleobase of the guide sequence and 12 nucleobases of the passenger region in the 5' to 3' direction. In this way, the guide sequence length remains 19 nucleosides, but the passenger sequence is shortened to 12 nucleosides.

[0216] In certain embodiments, if a third nucleoside region as described above exists, the third nucleoside region and optionally the 3'-terminal portion of the first nucleoside region (advantageously containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleosides) and / or the 5'-terminal portion of the second nucleoside region (advantageously containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleosides) can form a hairpin loop.

[0217] In certain embodiments, the hairpin loop contains 1 to 8, 2 to 7, 3 to 6 contiguous nucleosides, advantageously containing 4 or 5 contiguous nucleosides.

[0218] The oligomeric compounds of the first aspect disclosed herein can be blunt-ended. In the oligomeric compounds of the first aspect disclosed herein, the first or second nucleoside region may have overhangs.

[0219] In the oligomeric compounds of the first aspect disclosed herein, the first region can be selected from the sequences in Table 3a, or a portion thereof, particularly from the sequences of construct numbers 327, 352, 356, 362, 375, and 393.

[0220] In the oligomeric compounds of the first aspect disclosed herein, the second region may be selected from the sequences in Table 3b, or a portion thereof, particularly a portion of 14 nucleosides in length, and particularly selected from the sequences with construct numbers 427, 452, 456, 462, 475, and 493.

[0221] The total length of the oligomeric compound may be from about 25 to about 35 nucleosides, particularly about 33 or about 34 nucleosides.

[0222] In certain embodiments, the nucleobase of the terminal nucleoside at the 5'-end of the first region is selected from adenine (A), uracil (U), guanine (G), and cytosine (C), preferably U; and optionally, the terminal nucleoside at the 3'-end of the second region is replaced with a base complementary to the base at the 5'-end of the first region, preferably A.

[0223] Ligand

[0224] The oligomeric compound may comprise one or more ligands.

[0225] One or more ligands, particularly two or more, or three ligands, may be conjugated to the second linked nucleoside region and / or the first linked nucleoside region.

[0226] One or more ligands may be conjugated to the 3'-end region of the second linked nucleoside region and / or the first linked nucleoside region, preferably conjugated to the 3'-end nucleoside of the second linked nucleoside region and / or the first linked nucleoside region, and / or conjugated to the 5'-end nucleoside of the second linked nucleoside region. In particular, the ligand may be conjugated to the 3'-end nucleoside.

[0227] One or more ligands are any cell-targeting moieties, such as lipids, carbohydrates, aptamers, vitamins, and / or peptides that bind to specific targets on the cell membrane or cell surface.

[0228] One or more ligands may comprise one or more, particularly three, carbohydrates.

[0229] One or more, particularly three, carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0230] One or more carbohydrates may comprise or consist of one or more, particularly three, hexose moieties.

[0231] One or more, particularly three, hexose moieties are one or more galactose moieties, one or more lactose moieties, one or more (particularly three) N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0232] One or more carbohydrates may comprise one or more, particularly three, N - acetylgalactosamine moieties. Alternatively, one or more carbohydrates may comprise two or more N - acetylgalactosamine moieties, advantageously three.

[0233] One or more ligands are attached to the oligomeric compound in a linear configuration or a branched configuration, advantageously to its second linked nucleoside region.

[0234] Particularly advantageous ligands are as follows, also known as "toothbrushes":

[0235]

[0236] Without wishing to be bound by a particular theory, it is believed that due to the presence of such ligands, it is possible to selectively target the target tissue, namely the liver that produces angiotensinogen (AGT), so that the oligomeric compound can more effectively exert its inhibitory effect on the AGT gene.

[0237] One or more (particularly three) ligands may be attached to the oligomeric compound in a bi - antenna or tri - antenna configuration. The one or more ligands discussed above are advantageously attached to the 3'-terminal nucleoside of the second linked nucleoside region.

[0238] Internucleoside linkage

[0239] The oligomeric compounds of the first aspect disclosed herein may comprise internucleoside linkages, and at least one internucleoside linkage is a modified internucleoside linkage.

[0240] The modified internucleoside linkage may be a phosphorothioate or dithiophosphonate internucleoside linkage.

[0241] The oligomeric compounds of the first aspect disclosed herein may comprise 1 to 16 phosphorothioate or dithiophosphonate internucleoside linkages. Certain modified internucleoside linkages are the subject of certain subsequent embodiments. Certain modified internucleoside linkages are known in the art, for example, as described by Hu et al. in Signal Transduction and Targeted Therapy (2020) 5:101.

[0242] The oligomeric compound may comprise 7, 8, 9 or 10 phosphorothioate or dithiophosphonate internucleoside linkages. One or more phosphorothioate or dithiophosphonate internucleoside linkages may be present in the 5'-terminal region of the first linked nucleoside region. Advantageously, the oligomeric compound comprises three phosphorothioate internucleoside linkages at three adjacent nucleosides in the 5'-terminal region.

[0243] In addition, depending on the number of nucleosides present in the hairpin loop, the oligomeric compound may comprise phosphorothioate or phosphorodithioate internucleoside linkages between at least two, at least three, at least four, or at least five adjacent nucleosides in the hairpin loop. In particular, the oligomeric compound may comprise phosphorothioate or phosphorodithioate internucleoside linkages between each adjacent nucleoside present in the hairpin loop.

[0244] Modification

[0245] In the oligomeric compound according to the first aspect described above, at least one nucleoside may contain a modified sugar.

[0246] The modified sugar may be selected from 2'-modified sugars, conformationally restricted nucleoside (CRN) sugars (such as locked nucleic acid (LNA) sugars, (S)-constrained ethyl bicyclic nucleic acid, constrained ethyl (cEt) sugars), tricyclic DNA, morpholino, unlocked nucleic acid (UNA) sugars, glycol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA). Specific modified sugars are the subject of some of the subsequent exemplary embodiments. Certain modified sugars are known in the art, for example, as described by Hu et al. in Signal Transduction and Targeted Therapy (2020) 5:101.

[0247] The 2'-modified sugar may be selected from 2'-O-alkyl modified sugars, 2'-O-methyl modified sugars, 2'-O-methoxyethyl modified sugars, 2'-O-allyl modified sugars, 2'-C-allyl modified sugars, 2'-deoxy modified sugars (such as 2'-deoxyribose), 2'-F modified sugars, 2'-arabino furano fluoro modified sugars, 2'-O-benzyl modified sugars, and 2'-O-methyl-4-pyridyl modified sugars. At least one modified sugar may be a 2'-O-methyl modified sugar.

[0248] At least one modified sugar may be a 2'-F modified sugar, and advantageously, at most 16 or 17 sugars are 2'-F modified sugars. Advantageously, the sugar is ribose.

[0249] In the oligomeric compound of the first aspect disclosed herein, the sugar of the nucleoside at either the 2nd or 14th position downstream of the first nucleoside in the 5'-terminal region of the first linked nucleoside region does not contain a 2'-O-methyl modification. In certain embodiments, the 3'-terminal position of the second linked nucleoside region does not contain a 2'-O-methyl modification.

[0250] In certain embodiments, the sugar of the nucleoside at either the 2nd or 14th position downstream of the first nucleoside in the 5'-terminal region of the first linked nucleoside region contains a 2'-F modification.

[0251] In certain embodiments, among the nucleosides of the second linked nucleoside region, the sugars of those nucleosides corresponding to the nucleoside positions at any of the 11th to 13th positions downstream of the first nucleoside in the 5'-terminal region of the first linked nucleoside region contain a 2'-F modification.

[0252] In certain embodiments, the 3'-terminal nucleoside of the second linked nucleoside region contains a 2'-F modification.

[0253] In certain embodiments, one or more odd-numbered nucleosides starting from the 5'-terminal region of the first linked nucleoside region may be modified, and / or one or more even-numbered nucleosides starting from the 5'-terminal region of the first linked nucleoside region may be modified. Generally, the modification of the even-numbered nucleosides is a second modification different from the modification of the odd-numbered nucleosides.

[0254] In certain embodiments, one or more odd-numbered nucleosides starting from the 3'-terminal region of the second linked nucleoside region may be modified in a manner different from the modification of the odd-numbered nucleosides in the first linked nucleoside region.

[0255] In certain embodiments, one or more even-numbered nucleosides starting from the 3'-terminal region of the second linked nucleoside region are modified in a manner different from that of the even-numbered nucleosides in the first linked nucleoside region.

[0256] In certain embodiments, at least one or more modified even-numbered nucleosides in the first linked nucleoside region are adjacent to at least one or more odd-numbered nucleosides with a different modification pattern in the first nucleoside region.

[0257] In certain embodiments, at least one or more modified even-numbered nucleosides in the second linked nucleoside region are adjacent to at least one or more odd-numbered nucleosides with a different modification pattern in the second linked nucleoside region.

[0258] In certain embodiments, the sugars of one or more odd-numbered nucleosides starting from the 5'-terminal region of the first nucleoside region may be 2'-O-methyl modified sugars.

[0259] In certain embodiments, one or more even-numbered nucleosides starting from the 3'-terminal region of the first linked nucleoside region may be 2'-F modified sugars.

[0260] In certain embodiments, the sugars of one or more odd-numbered nucleosides starting from the 5'-terminal region of the second linked nucleoside region may be 2'-O-methyl modified sugars.

[0261] In certain embodiments, one or more even-numbered nucleosides starting from the 5'-terminal region of the second linked nucleoside region may be 2'-F modified sugars.

[0262] In certain embodiments, the sugars of multiple adjacent nucleosides in the first nucleoside region can be modified by the same or different modification methods.

[0263] In certain embodiments, the sugars of multiple adjacent nucleosides in the second nucleoside region can be modified by the same or different modification methods.

[0264] In certain embodiments, the sugars of multiple adjacent nucleosides in the hairpin loop can be modified by the same or different modification methods. The same modification can be a 2'-F modified sugar, or it can also be a 2'-O-methyl modified sugar.

[0265] Multiple adjacent 2'-O-methyl modified sugars can be present in at least eight adjacent nucleosides in the first and / or second nucleoside regions, or can also be present in three or four adjacent nucleosides in the hairpin loop.

[0266] In certain embodiments, as described above, the hairpin loop can include at least one nucleoside having a modified sugar.

[0267] In certain embodiments, at least one such nucleoside is adjacent to a nucleoside with a sugar having a different modification method. Advantageously, the sugar modification methods of all adjacent nucleosides in the hairpin loop are different. For example, the modified sugar is a 2'-O-methyl modified sugar, and the sugar with a different modification method is a 2'-F modified sugar.

[0268] In certain embodiments, one or more nucleosides in the first linked nucleoside region and / or the second linked nucleoside region can be inverted nucleosides, which are connected to the 3'-carbon of the sugar of an adjacent nucleoside through the 3'-carbon of its sugar; and / or one or more nucleosides in the first linked nucleoside region and / or the second linked nucleoside region are inverted nucleosides, which are connected to the 5'-carbon of the sugar of an adjacent nucleoside through the 5'-carbon of its sugar.

[0269] muRNA nucleic acid construct

[0270] In a second aspect, there is provided a nucleic acid construct having at least:

[0271] (a) A first nucleic acid portion that is at least partially complementary to at least a first portion of the RNA transcribed from the AGT gene;

[0272] (b) A second nucleic acid portion that is at least partially complementary to at least a second portion of the RNA transcribed from the AGT gene, the second portion being different from the first portion;

[0273] (c) A third nucleic acid portion that is at least partially complementary to the first nucleic acid portion in (a), thereby forming a first nucleic acid double-stranded region therewith;

[0274] (d) A fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion in (b) such that a second nucleic acid duplex region is formed therewith.

[0275] The construct can be designed to decompose after in vivo administration to produce at least first and second discrete nucleic acid targeting molecules that respectively target RNA portions transcribed from the target genes in (a) and (b). Among them, (i) the first nucleic acid targeting molecule is capable of regulating the expression of the target gene in (a) and comprises or is derived from at least the first nucleic acid portion in (a); (ii) the second nucleic acid targeting molecule is capable of regulating the expression of the target gene in (b) and comprises or is derived from the second nucleic acid portion in (b). The construct can also be designed such that after decomposition, the first and second discrete nucleic acid targeting molecules are respectively processed by independent RNA interference-induced silencing complexes.

[0276] The construct according to the second aspect and its foregoing embodiments comprises at least one labile function such that after in vivo administration, the construct is cleaved to produce at least first and second discrete nucleic acid targeting molecules.

[0277] The labile function can comprise one or more unmodified nucleotides. In particular, one or more unmodified nucleotides in the labile function represent one or more cleavage positions within the construct, and after in vivo administration, the construct is cleaved at these cleavage positions to produce at least first and second discrete nucleic acid targeting molecules. In particular, the cleavage positions can be respectively located within the construct such that after cleavage, the first discrete nucleic acid targeting molecule comprises or is derived from the first nucleic acid duplex region, and the second discrete nucleic acid targeting molecule comprises or is derived from the second nucleic acid duplex region. Advantageously, the first discrete nucleic acid targeting molecule comprises or consists of the first nucleic acid portion in (a) and the third nucleic acid portion in (c), and / or the second discrete nucleic acid targeting molecule comprises or consists of the second nucleic acid portion in (b) and the fourth nucleic acid portion in (d).

[0278] In certain embodiments:

[0279] (a) The first nucleic acid portion has a nucleobase sequence selected from SEQ ID NOs: 1 to 100 in Table 1a;

[0280] (b) The second nucleic acid portion: Its nucleobase sequence is selected from Table 1a (SEQ ID NOs: 1 to 100).

[0281] (c) The third nucleic acid portion: Its nucleobase sequence is selected from Table 1b (SEQ ID NOs: 101 to 200).

[0282] (d) The fourth nucleic acid portion: Its nucleobase sequence is selected from Table 1b (SEQ ID NOs: 101 to 200). If the lengths of the third and fourth nucleobase sequences are 14 nucleobases, their lengths can be reduced by 1, 2, or 3 nucleobases, and advantageously, the nucleobases at the 5' end are deleted.

[0283] In certain such embodiments, the first nucleic acid portion in (a) and the fourth nucleic acid portion in (d) may be directly or indirectly linked as a primary structure. In certain embodiments, the first and fourth nucleic acid portions may have nucleobase sequences of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141, 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and 200, 40 and 140, 75 and 175, 30 and 130, 42 and 142, 81 and 181, 17 and 117, 34 and 134, 53 and 153, 29 and 129, 26 and 126, 74 and 174, 94 and 194, 14 and 114, 3 and 103, 7 and 107, and 2 and 102, respectively. Advantageously, the sequence lengths of SEQ ID NOs: 27, 52, 56, 62, 75, and 93 may be reduced by 1, 2, 3, or 4 nucleobases, advantageously by deletion of nucleobases at the 5' end.

[0284] In certain embodiments, the second nucleic acid portion in (b) and the third nucleic acid portion in (c) may be directly or indirectly linked as a primary structure. In certain embodiments, the second and third nucleic acid portions may have nucleobase sequences of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141, 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and 200, 40 and 140, 75 and 175, 30 and 130, 42 and 142, 81 and 181, 17 and 117, 34 and 134, 53 and 153, 29 and 129, 26 and 126, 74 and 174, 94 and 194, 14 and 114, 3 and 103, 7 and 107, and 2 and 102, respectively. Advantageously, the sequence lengths of SEQ ID NOs: 27, 52, 56, 62, 75, and 93 may be reduced by 1, 2, 3, or 4 nucleobases, advantageously by deletion of nucleobases at the 5' end.

[0285] In certain embodiments, the construct may further comprise 1 to 8 additional nucleic acid portions, each of which is at least partially complementary to 1 to 8 additional RNA portions transcribed from one or more target genes. These target genes may be the same as or different from each other and / or the same as or different from the target genes defined in (a) and / or (b). Moreover, each of the 1 to 8 additional nucleic acid portions forms an additional double-stranded region with a respective transient nucleic acid portion to which it is at least partially complementary. In particular, the second nucleic acid portion in (b) and the 1 to 8 additional nucleic acid portions may be directly or indirectly linked to a selected transient nucleic acid portion as their respective primary structures.

[0286] In certain embodiments, a direct or indirect linkage may represent: (i) an internucleotide bond; (ii) an internucleotide gap; or (iii) a nucleic acid linker moiety consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, which is preferably single-stranded. Advantageously, the linkage may be direct, thereby forming a continuous strand.

[0287] In certain embodiments, there may be some complementarity between the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b), or between the third nucleic acid moiety in (c) and the fourth nucleic acid moiety in (d). Advantageously, such complementarity is manifested as:

[0288] (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base pairs may form, preferably 2, 3, 4, or 5 base pairs;

[0289] (ii) it may exist between the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b).

[0290] In certain embodiments, an internucleotide bond may involve at least one of one or more unmodified nucleotides, and preferably cleavage may occur at the 3'-position of the (at least one) unmodified nucleotide.

[0291] In certain embodiments, the lengths of the first nucleic acid moiety in (a), the second nucleic acid moiety in (b), the third nucleic acid moiety in (c), and / or the fourth nucleic acid moiety in (d) may be 7 to 25 nucleotides, respectively. Optionally, the lengths of the first nucleic acid moiety in (a) and / or the second nucleic acid moiety in (b) may be 18 to 21, 18 to 20, or 19 nucleotides. In a particular embodiment, the lengths of the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b) are 19 nucleotides. The lengths of the third nucleic acid moiety in (c) and / or the fourth nucleic acid moiety in (d) may be 11 to 20, 13 to 16, 14 or 15, or 14 nucleotides.

[0292] In certain embodiments, the lengths of the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b) are 19 nucleotides, and the lengths of the third nucleic acid moiety in (c) and the fourth nucleic acid moiety in (b) are 14 nucleotides.

[0293] In certain embodiments, unmodified nucleotides are located at positions 18 to 25, 18 to 21, and / or the 3'-terminal position of the first nucleic acid moiety in (a) and / or the third nucleic acid moiety in (c).

[0294] In certain embodiments, unmodified nucleotides are located at the 19th position.

[0295] In certain embodiments, the length of the first nucleic acid portion in (a) and the second nucleic acid portion in (b) is 19 nucleotides, the length of the third nucleic acid portion in (c) and the fourth nucleic acid portion in (b) is 14 nucleotides, and the unmodified nucleoside is located at the 19th position of the first nucleic acid portion in (a) and the second nucleic acid portion in (b).

[0296] In certain embodiments, the nucleic acid linking portion may be 1 to 8 nucleotides, 2 to 7 or 3 to 6 nucleotides, 4 or 5 or 4 nucleotides in length.

[0297] In certain embodiments, one or all of the double-stranded regions are independently 10 to 19, 13 to 19, 13, 14, or 15 base pairs in length, and optionally one mismatch may be present within the double-stranded region.

[0298] In certain embodiments, the nucleic acid construct can be blunt ended.

[0299] In certain embodiments, the first nucleic acid portion in (a); and / or

[0300] (b) the second nucleic acid portion; and / or

[0301] (c) the third nucleic acid portion; and / or

[0302] (d) the fourth nucleic acid portion; and / or

[0303] The 1 to 8 additional nucleic acid portions as defined above, if present, and the passenger nucleic acid portion as defined above, if present, may have overhangs.

[0304] In certain embodiments, the target RNA can be messenger RNA (mRNA) or other RNA molecules.

[0305] Ligand

[0306] The nucleic acid construct according to the second aspect and preceding embodiments thereof may further comprise one or more ligands.

[0307] In certain embodiments, the first nucleic acid portion in (a), the second nucleic acid portion in (b), the third nucleic acid portion in (c), the fourth nucleic acid portion in (d), and the 1 to 8 additional nucleic acid portions if present as defined above and the passenger nucleic acid portion as defined above may each have a 5' to 3' directionality, thereby determining their 5' and 3' regions.

[0308] In certain embodiments, one or more ligands are conjugated to the 3' region, advantageously the 3' end, of any of: (i) the third nucleic acid moiety in (c); and or (ii) the fourth nucleic acid moiety in (d); and (iii) the passenger nucleic acid moiety as defined above if present.

[0309] In certain embodiments, one or more ligands may be conjugated to one or more intermediate regions between the 5' and 3' regions of any nucleic acid moiety, advantageously to the intermediate region of the third nucleic acid moiety in (c), the fourth nucleic acid moiety in (d), and the passenger nucleic acid moiety as defined above.

[0310] In certain embodiments, one or more ligands may be conjugated to the 5' region of any nucleic acid moiety, advantageously to the 5' terminus.

[0311] In certain embodiments, one or more ligands may be any cell targeting moiety, such as a lipid, carbohydrate, aptamer, vitamin, and / or peptide that binds to a specific target on the cell membrane or cell surface. One or more carbohydrates may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. In one embodiment, one or more carbohydrates may comprise one or more hexose moieties, such as one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties. The hexose moiety may comprise two or three N-acetylgalactosamine moieties. In particular, the hexose moiety may comprise three N-acetylgalactosamine moieties.

[0312] In certain embodiments, one or more ligands may be linked in a linear configuration or a branched configuration. Advantageously, one or more ligands may be linked in a bi-antennary or tri-antennary configuration, or a configuration based on individual ligands at different positions.

[0313] Advantageously, the ligand may have the following structure:

[0314]

[0315] Internucleoside linkage

[0316] The nucleic acid construct as described in the second aspect above or its foregoing embodiments may comprise one or more phosphorothioate or phosphorodithioate internucleoside linkages.

[0317] In certain embodiments, the nucleic acid construct may comprise from 1 to 15 phosphorothioate or phosphorodithioate internucleoside linkages.

[0318] In certain embodiments, the nucleic acid construct may comprise one or more phosphorothioate or phosphorodithioate internucleoside linkages in one or more 5' and / or 3' regions of the following moieties: the first nucleic acid moiety in (a), the second nucleic acid moiety in (b), the third nucleic acid moiety in (c), the fourth nucleic acid moiety in (d), the 1 to 8 additional nucleic acid moieties as defined above, and the passenger nucleic acid moiety as defined above.

[0319] In certain embodiments, the nucleic acid construct may comprise a phosphorothioate or phosphorodithioate internucleotide linkage between at least two adjacent nucleotides of the nucleic acid linking moiety as defined above.

[0320] In certain embodiments, the nucleic acid construct may comprise a phosphorothioate or phosphorodithioate internucleotide linkage between each adjacent nucleotide where a nucleic acid linking moiety is present.

[0321] In certain embodiments, the nucleic acid construct may comprise phosphorothioate or phosphorodithioate internucleotide linkages linking the following moieties to a nucleic acid linking moiety as defined above:

[0322] (a) the first nucleic acid portion;

[0323] (b) the second nucleic acid portion;

[0324] (c) the third nucleic acid portion;

[0325] (d) the fourth nucleic acid portion;

[0326] 1 to 8 additional nucleic acid moieties as defined above;

[0327] The passenger nucleic acid moiety is as defined above.

[0328] Modification

[0329] In the nucleic acid construct described in the second aspect and the aforementioned embodiments thereof, at least one nucleotide in at least one of the following parts may be modified:

[0330] (a) the first nucleic acid portion;

[0331] (b) the second nucleic acid portion;

[0332] (c) the third nucleic acid portion;

[0333] (d) the fourth nucleic acid portion;

[0334] If present, 1 to 8 additional nucleic acid moieties as defined above;

[0335] If present, a passenger nucleic acid moiety as defined above;

[0336] If present, a nucleic acid linking moiety as further defined above.

[0337] In some embodiments, one or more odd-numbered nucleotides starting from the 5' region of a certain portion below can be modified, and / or one or more even-numbered nucleotides starting from the 5' region of a certain portion below can be modified, typically the modification of the even-numbered nucleotides is a second modification that is different from the modification of the odd-numbered nucleotides:

[0338] The first nucleic acid portion in (a);

[0339] The second nucleic acid portion in (b);

[0340] The third nucleic acid portion in (c);

[0341] The fourth nucleic acid portion in (d);

[0342] If present, one to eight additional nucleic acid portions as defined above;

[0343] If present, the passenger nucleic acid portion as defined above.

[0344] In certain embodiments:

[0345] One or more odd-numbered nucleotides starting from the 3' region of the third nucleic acid portion in (c) can be modified in a manner different from the modification of the odd-numbered nucleotides starting from the 5' region of the first nucleic acid portion in (a);

[0346] One or more odd-numbered nucleotides starting from the 3' region of the fourth nucleic acid portion in (d) can be modified in a manner different from the modification of the odd-numbered nucleotides starting from the 5' region of the second nucleic acid portion in (b);

[0347] If present, one or more odd-numbered nucleotides starting from the 3' region of the passenger nucleic acid portion as defined above can be modified in a manner different from the modification of the odd-numbered nucleotides starting from the 5' region of the one to eight additional nucleic acid portions as defined above;

[0348] If present, one or more nucleotides in the nucleic acid linking portion further defined above can be modified by: (i) being different from the modification of the nucleotide adjacent to the 3' region of the first nucleic acid portion in (a); and / or (ii) being different from the modification of the nucleotide adjacent to the 3' region of the second nucleic acid portion in (b); and / or being different from the modification of the nucleotide adjacent to the 3' region of the one to eight additional nucleic acid portions as defined above if present.

[0349] In certain embodiments, one or more even-numbered nucleotides starting from the 3' region of the following portions can be modified in a manner different from the modification of the odd-numbered nucleotides starting from the 3' region of these portions respectively:

[0350] (i) The third nucleic acid portion in (c);

[0351] (ii) The fourth nucleic acid portion in (d);

[0352] (iii) If present, the passenger nucleic acid portion as defined above.

[0353] In certain embodiments, at least one or more modified even-numbered nucleotides in (i) the first nucleic acid portion in (a), (ii) the second nucleic acid portion in (b), and (iii), if present, the 1 to 8 additional nucleic acid portions as defined above, may be adjacent to at least one or more odd-numbered nucleotides with a different modification pattern in each of these portions.

[0354] In certain embodiments, at least one or more modified even-numbered nucleotides in (i) the third nucleic acid portion in (c), (ii) the fourth nucleic acid portion in (d), and (iii), if present, the passenger nucleic acid portions as defined above, may be adjacent to at least one or more odd-numbered nucleotides with a different modification pattern in each of these portions.

[0355] In certain embodiments, multiple adjacent nucleotides in (i) the first nucleic acid portion in (a), (ii) the second nucleic acid portion in (b), and (iii), if present, the 1 to 8 additional nucleic acid portions as defined above, may be modified by a common modification pattern.

[0356] In certain embodiments, multiple adjacent nucleotides in (i) the third nucleic acid portion in (c), (ii) the fourth nucleic acid portion in (d), and (iii), if present, the passenger nucleic acid portions as defined above, may be modified by a common modification pattern.

[0357] In certain embodiments, the multiple adjacent, co-modified nucleotides may be 2 to 4 adjacent nucleotides, advantageously 3 or 4 adjacent nucleotides.

[0358] In certain embodiments, the multiple adjacent, co-modified nucleotides may be located in the 5' region of (i) the third nucleic acid portion in (c), (ii) the fourth nucleic acid portion in (d), and (iii), if present, the passenger nucleic acid portions as defined above.

[0359] In certain embodiments, the multiple adjacent, co-modified nucleotides may be located in the nucleic acid linking portion further defined above.

[0360] In certain embodiments:

[0361] (a) One or more modified nucleotides of the first nucleic acid portion may not have a common modification at the corresponding nucleotides of the third nucleic acid portion in the first double-stranded region (c);

[0362] (b) One or more modified nucleotides of the second nucleic acid portion may not have a common modification at the corresponding nucleotides of the fourth nucleic acid portion in the second double-stranded region (d);

[0363] If present, one or more modified nucleotides of the 1 to 8 additional nucleic acid moieties defined above may not have a common modification at the corresponding nucleotides of the respective passenger nucleic acid moieties in their respective duplex regions.

[0364] In certain embodiments:

[0365] (a) One or more modified nucleotides of the first nucleic acid moiety may be displaced by at least one nucleotide relative to the co-modified nucleotides of the third nucleic acid moiety in (c);

[0366] (b) One or more modified nucleotides of the second nucleic acid moiety may be displaced by at least one nucleotide relative to the co-modified nucleotides of the fourth nucleic acid moiety in (d);

[0367] If present, one or more modified nucleotides of the 1 to 8 additional nucleic acid moieties defined above may be displaced by at least one nucleotide relative to the co-modified nucleotides of the passenger nucleic acid moiety defined above, if present.

[0368] In certain embodiments, the modifications can each independently be a sugar modification, a phosphate modification, or a base modification.

[0369] In certain embodiments, the modifications can be selected from nucleotides having a 2'-modified sugar; conformationally restricted nucleotide (CRN) sugars such as locked nucleic acid (LNA), (S)-constrained ethyl bicyclic nucleic acid, constrained ethyl (cEt), tricyclic DNA; morpholino, unlocked nucleic acid (UNA), glycol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA). The 2'-modified sugars can be selected from 2'-O-alkyl-modified sugars, 2'-O-methyl-modified sugars, 2'-O-methoxyethyl-modified sugars, 2'-O-allyl-modified sugars, 2'-C-allyl-modified sugars, 2'-deoxy-modified sugars (such as 2'-deoxyribose), 2'-F-modified sugars, 2'-arabino-furanosyl fluoride-modified sugars, 2'-O-benzyl-modified sugars, 2'-amino-modified sugars, and 2'-O-methyl-4-pyridyl-modified sugars.

[0370] In certain embodiments, the base modification can be either an abasic nucleotide or a nucleotide containing a non-natural base.

[0371] In certain embodiments, at least one modification can be a 2'-O-methyl modification of the ribose moiety.

[0372] In certain embodiments, at least one modification can be a 2'-F modification of the ribose moiety.

[0373] In certain embodiments, the ribose moiety of the nucleotide at either the 2nd or 14th position downstream of the first nucleotide in the 5' region of (i) the first nucleic acid moiety in (a), (ii) the second nucleic acid moiety in (b), and (iii) if present, any one of the 1 to 8 additional nucleic acid moieties as defined above may not contain a 2'-O-methyl modification.

[0374] In certain embodiments, in (i) the third nucleic acid moiety in (c), (ii) the fourth nucleic acid moiety in (d), and (iii) if present, the passenger nucleic acid moiety as defined above, one, two, or all three nucleotides corresponding to the nucleotide at any one of the 11th to 13th positions downstream of the first nucleotide in the 5' region of (i) the first nucleic acid moiety in (a), (ii) the second nucleic acid moiety in (b), and (iii) if present, any one of the 1 to 8 additional nucleic acid moieties as defined above may not contain a 2'-O-methyl modification.

[0375] In certain embodiments, the ribose moiety of the nucleotide at either the 2nd or 14th position downstream of the first nucleotide in the 5' region of (i) the first nucleic acid moiety in (a), (ii) the second nucleic acid moiety in (b), and (iii) if present, any one of the 1 to 8 additional nucleic acid moieties as defined above may contain a 2'-F modification.

[0376] In certain embodiments, in (i) the third nucleic acid moiety in (c), (ii) the fourth nucleic acid moiety in (d), and (iii) if present, the passenger nucleic acid moiety as defined above,

[0377] one, two, or all three nucleotides corresponding to the nucleotide at any one of the 11th to 13th positions downstream of the first nucleotide in the 5' region of (i) the first nucleic acid moiety in (a), (ii) the second nucleic acid moiety in (b), and (iii) if present, any one of the 1 to 8 additional nucleic acid moieties as defined above may contain a 2'-F modification

[0378] In certain embodiments, except for the unmodified nucleotides in the labile linkages as defined herein, the ribose moieties of all the remaining nucleotides may contain a 2'-O-methyl modification or a 2'-F modification. Advantageously, the ribose moieties of the remaining nucleotides may contain a 2'-O-methyl modification.

[0379] In certain embodiments, one or more (advantageously one) unmodified nucleotides represent any nucleotide in the nucleic acid linkage moiety further defined above, advantageously the nucleotide adjacent to the following in the nucleic acid linkage moiety further defined above: (i) the third nucleic acid moiety in (c); and / or (ii) the fourth nucleic acid moiety in (d); and / or (iii) if present

[0380] In the passenger nucleic acid portion as defined above.

[0381] In certain embodiments:

[0382] (a) The first nucleic acid portion can be selected from Table 3a;

[0383] (b) The second nucleic acid portion can be selected from Table 3a;

[0384] (c) The third nucleic acid portion can be selected from Table 3b;

[0385] (d) The fourth nucleic acid portion can be selected from Table 3b. The first nucleic acid portion and the second nucleic acid portion can be selected from Table 3a, and the first and second nucleic acid portions are different; the third and fourth nucleic acid portions can be selected from Table 3b.

[0386] In certain embodiments, the 3'-terminal positions of the first and third nucleic acid portions can be replaced with unmodified nucleotides.

[0387] In certain embodiments, the nucleic acid construct can comprise at least one vinyl phosphonate modification, such as comprising at least one vinyl phosphonate modification in the 5'-region of the following portions: (i) the first nucleic acid portion in (a); and / or (ii) the second nucleic acid portion in (b); and / or (iii) if present, 1 to 8 additional nucleic acid portions as defined above.

[0388] In certain embodiments, one or more nucleotides in the following portions can be inverted nucleotides, which can be linked to the 3'-carbon of an adjacent nucleotide through the 3'-carbon of the nucleotide, and / or can be an inverted nucleotide, through the 5'-carbon of the nucleotide

[0389] linked to the 5'-carbon of an adjacent nucleotide:

[0390] The first nucleic acid portion in (a);

[0391] The second nucleic acid portion in (b);

[0392] The third nucleic acid portion in (c);

[0393] The fourth nucleic acid portion in (d);

[0394] If present, 1 to 8 additional nucleic acid portions as defined above;

[0395] If present, the passenger nucleic acid portion as defined above.

[0396] In certain embodiments, the inverted nucleotide can be linked to an adjacent nucleotide through a phosphate group via a phosphodiester bond;

[0397] Alternatively, it can be linked to an adjacent nucleotide via a phosphorothioate group; or it can be linked to an adjacent nucleotide via a dithiophosphate group.

[0398] Different target genes

[0399] (b) The second nucleic acid moiety is at least partially complementary to at least a second part of the RNA transcribed from one target gene, which is different from the AGT gene. This can in particular simultaneously treat diseases or disorders related to AGT, as well as other disorders related to different targets described below.

[0400] Target genes different from the AGT gene can be selected from the following group:

[0401] · Genes encoding atherogenic lipoproteins containing apolipoprotein B (ApoB);

[0402] · Genes encoding proteins related to the inflammatory signaling pathway, such as interleukin-6 (IL-6), C-reactive protein (CRP), and interleukin-11 (IL-11);

[0403]

[0404] · Genes encoding other proteins related to hypertension;

[0405] · Genes encoding proteins related to platelet aggregation and the coagulation pathway;

[0406] · Genes encoding proteins related to diabetes;

[0407] · Genes encoding proteins related to obesity and metabolic syndrome; · Genes encoding proteins related to other atherogenic factors and modifiable risk factors for cardiovascular disease.

[0408] In particular, target genes different from the AGT gene are selected from the following group:

[0409] · APOC3 gene;

[0410] · PCSK9 gene;

[0411] · ANGPTL3 gene;

[0412] · ANGPTL4 gene;

[0413] · Lp(a) gene;

[0414] · ANGPTL 8 gene;

[0415] · ASGR1 / 2 gene;

[0416] · Advantageously, the APOC3 gene or the PCSK9 gene.

[0417] Multi-target microRNA (muRNA) of AGT and APOC3

[0418] Thus, in certain embodiments, a target gene different from the AGT gene can be the APOC3 gene. In this particular case, (b) the second nucleic acid portion is the APOC3 antisense strand, and (d) the fourth nucleic acid portion is a strand that is at least partially complementary to it.

[0419] Specifically:

[0420] (a) The first nucleic acid portion is selected from Table 1a;

[0421] (b) The second nucleic acid portion is selected from Table 4a;

[0422] (c) The third nucleic acid portion is selected from Table 1b;

[0423] (d) The fourth nucleic acid portion is selected from Table 4c.

[0424] In addition or alternatively,

[0425] (a) The first nucleic acid portion is selected from Table 3a;

[0426] (b) The second nucleic acid portion is selected from Table 4b;

[0427] (c) The third nucleic acid portion is selected from Table 3b;

[0428] (d) The fourth nucleic acid portion is selected from Table 4d.

[0429] In addition or alternatively,

[0430] (a) The first nucleic acid portion is selected from Table 1a;

[0431] (b) The second nucleic acid portion is selected from Table 5a;

[0432] (c) The third nucleic acid portion is selected from Table 1b;

[0433] (d) The fourth nucleic acid portion is selected from Table 5c.

[0434] In addition or alternatively,

[0435] (a) The first nucleic acid portion is selected from Table 3a;

[0436] (b) The second nucleic acid portion is selected from Table 5b;

[0437] (c) The third nucleic acid portion is selected from Table 3b;

[0438] (d) The fourth nucleic acid portion is selected from Table 5d.

[0439] In addition or alternatively,

[0440] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0441] (b) The second nucleic acid portion is selected from Table 10b, particularly Table 12b;

[0442] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0443] (d) The fourth nucleic acid portion is selected from Table 11b, particularly Table 13b.

[0444] Advantageously, regardless of whether (a) the first nucleic acid portion is selected from the unmodified form of Table 1a or the modified form of Table 3a, it is selected from the group of constructs labeled AGT_27, AGT_62, AGT_52, AGT_56, AGT_93, and AGT_75. In this embodiment, the corresponding (c) third nucleic acid portion has a corresponding label in Table 1b or Table 3b.

[0445] Furthermore, advantageously, (b) the second nucleic acid portion is selected from the group consisting of A28(14-4)mF and A277(12-5) in Table 6a (particularly Table 6b), and the second portion consists of the first 19 nucleotides of these entries. The corresponding (d) fourth portion contains the remaining nucleotides of the corresponding entry in Table 6a (particularly Table 6b).

[0446] The nucleic acid portions targeting the AGT gene can be arbitrarily combined with the nucleic acid portions targeting the APOC3 gene described above, such as AGT_27 + A28(14-4)mF, AGT_27 + A277(12-5), AGT_62 + A28(14-4)mF, etc., thereby obtaining multi-target microRNAs (muRNA) that simultaneously target AGT and APOC3. Due to the activities of the compounds described in the examples, such combinations are also reasonable.

[0447] Therefore, diseases or disorders related to AGT and diseases or disorders related to APOC3 can be treated simultaneously. Multi-target microRNAs (muRNA) of AGT and PCSK9

[0448] In certain embodiments, the target gene different from the AGT gene may be the PCSK9 gene. In this particular case, (b) the second nucleic acid portion is, for example, the PCSK9 antisense strand, and (d) the fourth nucleic acid portion is a strand that is at least partially complementary to it.

[0449] Furthermore or alternatively,

[0450] (a) The first nucleic acid portion is selected from Table 3a;

[0451] (b) The second nucleic acid portion is selected from Table 7a;

[0452] (c) The third nucleic acid portion is selected from Table 3b;

[0453] (d) The fourth nucleic acid portion is selected from Table 7b.

[0454] Additionally or alternatively,

[0455] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0456] (b) The second nucleic acid portion is selected from Table 8a, particularly Table 8b;

[0457] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0458] (d) The fourth nucleic acid portion is selected from Table 8c, particularly Table 8d.

[0459] Additionally or alternatively,

[0460] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0461] (b) The second nucleic acid portion is selected from Table 9a, particularly Table 9b;

[0462] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0463] (d) The fourth nucleic acid portion is selected from Table 9c, particularly Table 9d.

[0464] Additionally or alternatively,

[0465] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0466] (b) The second nucleic acid portion is selected from Table 10a, particularly Table 11a;

[0467] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0468] (d) The fourth nucleic acid portion is selected from Table 12a, particularly Table 13a.

[0469] Particularly advantageously, regardless of whether (a) the first nucleic acid portion is selected from the unmodified form of Table 1a or the modified form of Table 3a, it is selected from the group of constructs labeled AGT_27, AGT_62, AGT_52, AGT_56, AGT_93, and AGT_75. In this particularly advantageous embodiment, the corresponding (c) third nucleic acid portion has a corresponding label in Table 1b or Table 3b.

[0470] In addition, it is particularly advantageous that (b) the second nucleic acid portion is selected from the group consisting of PC S29, PC S44, and PC S53 in Table 22, and the second portion consists of the first 19 nucleotides of these entries. The corresponding (d) fourth portion contains the remaining nucleotides of the corresponding entries in Table 22.

[0471] The nucleic acid portions targeting the AGT gene can be combined arbitrarily with the nucleic acid portions targeting the PCSK9 gene described above, such as AGT_27 + PCS29, AGT_27 + PCS44, AGT_27 + PCS53, AGT_62 + PCS29, etc., thereby obtaining multi-target microRNA (muRNA) molecules that simultaneously target AGT and PCSK9. Due to the activities of the various compounds described in the examples, such combinations are also reasonable. Therefore, diseases or disorders related to AGT and diseases or disorders related to PCSK9 can be treated simultaneously.

[0472] Multi-target microRNA (muRNA) of AGT / APOC3 and PCSK9

[0473] In certain embodiments, the construct further comprises 1 to 8 additional nucleic acid portions, each of which is at least partially complementary to 1 to 8 additional RNA portions transcribed from one or more target genes, which target genes are different from each other and / or the same or different from the target genes defined in (a) and (b). Moreover, each of the 1 to 8 additional nucleic acid portions forms an additional double-stranded region with a respective passenger nucleic acid portion that is at least partially complementary thereto. Advantageously, the construct comprises 1 additional nucleic acid portion.

[0474] In certain embodiments, the construct targets target genes selected from the following group:

[0475] In certain embodiments, the combinations of target genes targeted by the construct are as follows:

[0476] (a) the AGT gene, (b) the APOC3 gene, and (e) the PCSK9 gene;

[0477] (a) the AGT gene, (b) the ANGPTL3 gene, and (e) the Lp(a) gene;

[0478] (a) the AGT gene, (b) the APOC3 gene, and (e) the Lp(a) gene.

[0479] Among them, (a) is the first nucleic acid portion that is at least partially complementary to at least the first portion of the RNA transcribed from the AGT gene; (b) is the second nucleic acid portion that is at least partially complementary to the first portion of the RNA transcribed from the second gene; (e) is the fifth nucleic acid portion that is at least partially complementary to the third portion of the RNA transcribed from the third gene; (f) is the sixth nucleic acid portion that is at least partially complementary to the (e) portion. Advantageously, the target genes are (a) the AGT gene, (b) the APOC3 gene, and (e) the PCSK9 gene.

[0480] In certain embodiments, the target genes are (a) the AGT gene, (b) the APOC3 gene, and (e) the PCSK9 gene.

[0481] Accordingly,

[0482] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0483] (b) The second nucleic acid portion is selected from Table 4a;

[0484] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0485] (d) The fourth nucleic acid portion is selected from Table 4c;

[0486] (e) The fifth nucleic acid portion is selected from Table 7a;

[0487] (f) The sixth nucleic acid portion is selected from Table 7b.

[0488] In addition or alternatively,

[0489] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0490] (b) The second nucleic acid portion is selected from Table 4a, particularly Table 4b;

[0491] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0492] (d) The fourth nucleic acid portion is selected from Table 4c, particularly Table 4d;

[0493] (e) The fifth nucleic acid portion is selected from Table 8a, particularly Table 8b;

[0494] (f) The sixth nucleic acid portion is selected from Table 8c, particularly Table 8d.

[0495] In addition or alternatively,

[0496] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0497] (b) The second nucleic acid portion is selected from Table 4a, particularly Table 4b;

[0498] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0499] (d) The fourth nucleic acid portion is selected from Table 4c, particularly Table 4d;

[0500] (e) The fifth nucleic acid portion is selected from Table 8a, particularly Table 8b;

[0501] (f) The sixth nucleic acid portion is selected from Table 8c, particularly Table 8d.

[0502] In addition or alternatively,

[0503] (a) The first nucleic acid portion is selected from Table 1a;

[0504] (b) The second nucleic acid portion is selected from Table 5a;

[0505] (c) The third nucleic acid portion is selected from Table 1b;

[0506] (d) The fourth nucleic acid portion is selected from Table 5c;

[0507] (e) The fifth nucleic acid portion is selected from Table 9a, particularly Table 9b;

[0508] (f) The sixth nucleic acid portion is selected from Table 9c, particularly Table 9d.

[0509] In addition or alternatively,

[0510] (a) The first nucleic acid portion is selected from Table 3a;

[0511] (b) The second nucleic acid portion is selected from Table 5b;

[0512] (c) The third nucleic acid portion is selected from Table 3b;

[0513] (d) The fourth nucleic acid portion is selected from Table 5d;

[0514] (e) The fifth nucleic acid portion is selected from Table 9a, particularly Table 9b;

[0515] (f) The sixth nucleic acid portion is selected from Table 9c, particularly Table 9d.

[0516] In addition or alternatively,

[0517] (a) The first nucleic acid portion is selected from Table 1a, particularly Table 3a;

[0518] (b) The second nucleic acid portion is selected from Table 10b, particularly Table 12b;

[0519] (c) The third nucleic acid portion is selected from Table 1b, particularly Table 3b;

[0520] (d) The fourth nucleic acid moiety is selected from Table 11b, in particular Table 13b;

[0521] (e) The fifth nucleic acid moiety is selected from Table 10b, in particular Table 11b;

[0522] (f) The sixth nucleic acid moiety is selected from Table 12b, in particular Table 12b.

[0523] Certain advantageous nucleic acid moieties are listed under the headings “Multi-target microRNA (muRNA) of AGT and APOC3” and “Multi-target microRNA (muRNA) of AGT and PCSK9”. Among these nucleic acid moieties, all combinations are possible, such as AGT_27 + A28(14-4)mF + PCS44, AGT_62 + 277(12-5) + PCS29, provided that these specific constructs contain or consist of a construct targeting AGT, a construct targeting APOC3, and a construct targeting PCSK9. Due to the activities of the compounds described in the examples, such combinations are also feasible. Thus, diseases or disorders related to AGT, diseases or disorders related to APOC3, and diseases or disorders related to PCSK9 can be treated simultaneously.

[0524] Compositions and pharmaceutical compositions comprising shRNA, mxRNA, and / or muRNA oligomeric constructs

[0525] In a third aspect, the composition comprises the oligomeric compound of the first aspect and / or the nucleic acid construct of the second aspect, and a physiologically acceptable excipient.

[0526] According to a fourth aspect, there is provided a pharmaceutical composition comprising the oligomeric compound of the first aspect and / or the nucleic acid construct of the second aspect.

[0527] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, antioxidant, and / or preservative.

[0528] The oligomeric compound of the first aspect and / or the construct of the second aspect can be the sole pharmaceutically active ingredient.

[0529] Alternatively, the pharmaceutical composition further comprises one or more other pharmaceutically active ingredients. The other pharmaceutically active ingredients may be drugs for reducing hypertension and may be selected from diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, β-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists, non-selective α-adrenergic antagonists, synthetic steroidal mineralocorticoid drugs; combinations of any of the above drugs; and antihypertensive drugs formulated as fixed-dose combinations. More preferably, it is an angiotensin II receptor antagonist selected from losartan, valsartan, olmesartan, eprosartan, and azilsartan. Advantageously, the oligomeric compound and / or nucleic acid construct and the other pharmaceutically active ingredients may be administered simultaneously or in any order.

[0530] Diseases and other uses treatable with shRNA, mxRNA, and / or muRNA oligomeric compounds

[0531] According to the fifth aspect, there is provided the oligomeric compound of the first aspect and / or the nucleic acid construct of the second aspect, for use in human or veterinary medicine or therapy.

[0532] In a sixth aspect, the oligomeric compound of the first aspect and / or the nucleic acid construct of the second aspect may be used for treating, ameliorating, and / or preventing a disease or disorder.

[0533] One such disease may be an AGT-related disease or disorder discussed below. However, more diseases can be treated simultaneously using the muRNA constructs described herein. These diseases may for example be diseases or disorders associated with apolipoprotein B (ApoB)-containing atherogenic lipoproteins; diseases associated with inflammatory signaling pathways, such as diseases associated with interleukin-6 (IL-6), C-reactive protein (CRP), and interleukin-11 (IL-11); diseases associated with platelet aggregation and coagulation pathways; diabetes; obesity and metabolic syndrome; and / or diseases caused by other atherogenic factors and modifiable risk factors associated with cardiovascular diseases.

[0534] The corresponding targets, particularly in terms of dyslipidemia, may be selected from the group consisting of apolipoprotein C3 (APOC3), proprotein convertase subtilisin / kexin type 9 (PCSK9), angiopoietin-like protein 3 (ANGPTL3), angiopoietin-like protein 4 (ANGPTL4), lipoprotein(a) [Lp(a)], angiopoietin-like protein 8 (ANGPTL8), and asialoglycoprotein receptor 1 / 2 (ASGR1 / 2).

[0535] AGT-related diseases or disorders

[0536] The disease or disorder may be a disease or disorder associated with AGT, or a disease or disorder that requires reduction of AGT expression.

[0537] Specifically, the disease or disorder is selected from the following group: hypertension, hypertensive disease, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension (renal ischemic hypertension), increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular lesions, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypotension, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, fetal growth restriction (IUGR), fetal growth retardation, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes and metabolic syndrome.

[0538] APOC3-related disease or disorder

[0539] In addition to the diseases or disorders discussed previously, the disease or disorder may also be a disease or disorder associated with APOC3, or a disease or disorder that requires reduction of APOC3 expression level. Such diseases or disorders are preferably selected from: dyslipidemia (including mixed dyslipidemia); hyperchylomicronemia (including familial hyperchylomicronemia); hypertriglyceridemia (preferably severe hypertriglyceridemia and / or hypertriglyceridemia with a blood triglyceride level higher than 500 mg / dl); inflammation (including mild inflammation); atherosclerosis; atherosclerotic cardiovascular disease (ASCVD, including major adverse cardiovascular events such as myocardial infarction, stroke and peripheral arterial disease); pancreatitis (including acute pancreatitis).

[0540] PCSK9-related disease or disorder

[0541] In addition to the diseases or disorders discussed previously, the disease or disorder may also be a disease or disorder associated with PCSK9, or a disease or disorder that requires reduction of low density lipoprotein (LDL) cholesterol. Such diseases or disorders are preferably selected from: dyslipidemia (including mixed dyslipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia); atherosclerosis; atherosclerotic cardiovascular disease (ASCVD, including myocardial infarction, stroke and peripheral arterial disease).

[0542] The seventh aspect provides a method for treating a disease or disorder by administering to an individual in need thereof the oligomeric compound described in the first aspect and / or the nucleic acid construct described in the second aspect. The oligomeric compound and / or the nucleic acid construct can be administered to the individual by subcutaneous or intravenous injection.

[0543] According to the eighth aspect, the oligomeric compound described in the first aspect or the nucleic acid construct described in the second aspect can be used as a gene function analysis tool in research.

[0544] According to the ninth aspect, the oligomeric compound described in the first aspect and / or the nucleic acid construct described in the second aspect can be used to prepare a medicament for treating a disease or disorder.

[0545] Constructs and sequences of oligomeric compounds

[0546] The following table shows the nucleobase sequences of the antisense and sense strands of the oligomeric compound, the nucleobase sequence of the single-stranded oligomeric compound, and the definitions of the modified oligomeric compound. The relevant notations include the nucleobase sequence, sugar modification, and (where applicable) modified phosphate group.

[0547] The notations used are common in the art and have the following meanings:

[0548] A represents adenine;

[0549] U represents uracil;

[0550] C represents cytosine;

[0551] G represents guanine.

[0552] 5Phos represents a 5'-terminal phosphate group, which is advantageous but not essential;

[0553] m represents a 2'-O-methyl modification of the nucleoside sugar;

[0554] f represents a 2'-fluoro modification of the nucleoside sugar;

[0555] r represents an unmodified (2'-OH) ribonucleotide;

[0556] [Ps] or # represents a phosphorothioate internucleoside linkage;

[0557] i represents an inverted internucleoside linkage, which can be either a 3'-3' linkage or a 5'-5' linkage;

[0558] 3xGalNAc represents trivalent N-acetylgalactosamine (GalNAc).

[0559] Tables 1a and 1b below show the nucleobase sequences of the antisense and sense strands of 100 oligomeric compounds according to the examples.

[0560] Content of Table 1a

[0561] Table 1a: Nucleobase Sequences of the Antisense Strands of 100 Constructs

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569] Table 1b: Nucleobase Sequences of the Sense Strands of 100 Constructs

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576] Note: In particular, the nucleobase of the nucleotide at the 3'-end of each sense strand presented in the table can be replaced by adenine (A).

[0577] The following Table 2 shows the nucleobase sequences of 100 hairpin structure constructs selected according to the examples. These nucleobase sequences are the direct fusions of the antisense sequences in Table 1a and the corresponding sense sequences in Table 1b.

[0578] Table 2: Nucleobase Sequences of 100 Constructs by Combining the Sense and Antisense Sequences in Table 1a and Table 1b.

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585] Note: In particular, the nucleobase of the nucleotide at the 3' end of each sense strand presented in the table can be replaced by adenine (A).

[0586] The following Tables 3a to 3c respectively show 100 antisense sequences, sense sequences, and hairpin structure sequences, with complete modification information (modified sugar groups, and modified phosphate groups where applicable).

[0587] Table 3a: Modified antisense constructs

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597] Table 3b: Modified sense strand constructs

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604] Note: The 3'-end group of each of the above constructs may or may not be linked to "triple N-acetylgalactosamine (3xGalNAc)". Advantageously, these constructs bear a triple N-acetylgalactosamine ligand, particularly the toothbrush-type ligand as defined herein.

[0605] Table 3c: Modified hairpin structure constructs

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612] Note: The 5'-end group of each of the above constructs may or may not be phosphorylated. Additionally, independently, the 3'-end group of each of the above constructs may or may not be linked to "triple N-acetylgalactosamine (3xGalNAc)". Advantageously, these constructs bear a triple N-acetylgalactosamine ligand, particularly the toothbrush-type ligand as defined herein. Although a 5'-end phosphate is not strictly required, it is particularly advantageous for the constructs to bear a 5'-end phosphate because mammalian cells will add it themselves if it is absent in the administered molecule.

[0613] Specific explanations regarding the nomenclature in Tables 3a to 3c:

[0614] fN: 2'-fluoro residue

[0615] mN: 2'-O-methyl residue

[0616] Ps: phosphorothioate

[0617] p, Phos: phosphate

[0618] (GalNAc): Sirnaomics' mono-N-acetylgalactosamine building block

[0619] In addition, when using a notation such as "A277(12-5)mF", "A277" represents a sequence applicable to apolipoprotein C3 (APOC3) RNA interference (RNAi). The first number in the parentheses (in this case, 12) represents the number of base pairs in the short hairpin RNA (shRNA) double-stranded region, and the second number in the parentheses (in this case, 5) represents the number of nucleotides in the shRNA hairpin loop. If there is no annotation after the hyphen in the parentheses, it means the loop consists of 5 nucleotides.

[0620] The following Tables 4a to 4d show the nucleobase sequences of the antisense and sense strands of 376 APOC3 constructs screened according to the examples, as well as the sugar-phosphate backbone modifications. The 30 specific oligomeric compounds disclosed were screened from these 37 constructs.

[0621] Table 4a: Nucleobase sequences of the antisense strands of apolipoprotein C3 (APOC3) for 376 constructs

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632] Table 4b: Nucleobase sequences of the antisense strands of apolipoprotein C3 (APOC3) for 376 constructs and sugar-phosphate backbone modifications

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643] Table 4c: Nuclear base sequence of the sense strand of apolipoprotein C3 (APOC3) for 376 constructs

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654] Table 4d: Nuclear base sequence of the sense strand of apolipoprotein C3 (APOC3) for 376 constructs and sugar-phosphate backbone modification status

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665] The following Tables 5 to 5d show the nucleobase sequences of the antisense and sense strands of another 15 constructs and the sugar-phosphate backbone modifications.

[0666] Table 5a: Nucleobase sequences of the antisense strands of apolipoprotein C3 (APOC3) of another 15 constructs

[0667] Sequence number Unmodified antisense strand nucleobase sequence 2105 UAACUCAGAGAACUUGUCC 2106 UUGUCCUUAACGGUGCUCC 2107 UAAUCCCAGAACUCAGAGA 2108 UCCUUGGCGGUCUUGGUGG 2109 UCUGAAGCCAUCGGUCACC 2110 UCAGAGAACUUGUCCUUAA 2111 UACUCAGAGAACUUGUCCU 2112 UGAACUCAGAGAACUUGUC 2113 UACUUGUCCUUAACGGUGC 2114 UCUCAGAGAACUUGUCCUU 2115 UUUGUCCUUAACGGUGCUC 2116 UUCCUUGGCGGUCUUGGUG 2117 UGCUCCAGUAGUCUUUCAG 2118 UCAUCCUCGGCCUCUGAAG 2119 UUGGUGGCGUGCUUCAUGU

[0668] Table 5b: Nucleobase sequences of the antisense strands of apolipoprotein C3 (APOC3) of another 15 constructs and sugar-phosphate backbone modifications

[0669]

[0670]

[0671] Table 5c: Nucleobase sequences of the sense strands of apolipoprotein C3 (APOC3) of another 15 constructs

[0672]

[0673]

[0674] Table 5d: Nucleobase sequences of the sense strands of another 15 constructs and sugar-phosphate backbone modifications

[0675]

[0676] The following Tables 6a to 6b show the nucleobase sequences and sugar-phosphate backbone modifications of another 12 apolipoprotein C3 (APOC3) constructs.

[0677] Table 6a: Nucleobase sequences of each strand of another 12 apolipoprotein C3 (APOC3) constructs

[0678] Sequence number Experiment label Unmodified strand (5' to 3') 2165 A277(15) uuggauaggc agguggacuc accugccuau ccaa 2166 A28(15) ucaacaagga guacccgggg guacuccuug uuga 2167 A277(14) uuggauaggc agguggacua ccugccuauc caa 2168 A28(14) ucaacaagga guacccgggg uacuccuugu uga 2169 A277(12-5) uuggauaggc agguggacug ccuauccaa 2170 A277(13-4) uuggauaggc agguggacuu gccuauccaa 2171 A28(14-4) ucaacaagga guacccgggu acuccuuguu ga 2172 A277(14)mF uuggauaggc agguggacua ccugccuauc caa 2173 A28(14)mF ucaacaagga guacccgggg uacuccuugu uga 2174 A277(12-5)mF uuggauaggc agguggacug ccuauccaa 2175 A277(13-4)mF uuggauaggc agguggacuu gccuauccaa 2176 A28(14-4)mF ucaacaagga guacccgggu acuccuuguu ga

[0679] Table 6b: Nucleobase sequences of each strand of another 12 apolipoprotein C3 (APOC3) constructs and sugar-phosphate backbone modifications

[0680]

[0681]

[0682] In Example 9 below, the nucleobase sequences of the antisense and sense strands of 27 specific oligomeric compounds targeting proprotein convertase subtilisin / kexin type 9 (PCSK9) are given. These sequences are also the subject of specific embodiments further disclosed herein.

[0683] Tables 7a and 7b below show the specific sugar - phosphate backbone modifications of the PCSK9 antisense and sense strands of these 27 constructs.

[0684] Table 7a: Exemplary sugar - phosphate backbone modifications of the PCSK9 antisense strands of 27 constructs.

[0685]

[0686]

[0687]

[0688] Table 7b: Exemplary sugar - phosphate backbone modifications of the PCSK9 sense strands of 27 constructs.

[0689]

[0690]

[0691] Tables 8a to 8d below show the nucleobase sequences and sugar - phosphate backbone modifications of the proprotein convertase subtilisin / kexin type 9 (PCSK9) antisense and sense strands of 250 constructs selected according to Example 8 below. The 27 PCSK9 oligomeric compounds disclosed above were selected from these 250 constructs.

[0692] Table 8a: Nucleobase sequences of 250 unmodified PCSK9 antisense strands

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700] Table 8b: 250 modified PCSK9 antisense strand nucleobase sequences corresponding to Table 8a.

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709] Table 8c: 250 unmodified PCSK9 sense strand nucleobase sequences corresponding to Table 8a.

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717] Table 8d: 250 modified PCSK9 sense strands corresponding to the antisense strands in Table 8a.

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725] The following Tables 9a to 9d show the nucleobase sequences of the antisense and sense strands of proprotein convertase subtilisin / kexin type 9 (PCSK9) and the sugar-phosphate backbone modifications for an additional 43 constructs.

[0726] Table 9a: Nucleobase sequences of 43 unmodified PCSK9 antisense strands.

[0727]

[0728]

[0729] Table 9b: Nucleobase sequences of 43 modified PCSK9 antisense strands

[0730]

[0731]

[0732]

[0733] Table 9c: Nucleobase sequences of 43 unmodified PCSK9 sense strands corresponding to Table 9a.

[0734]

[0735]

[0736] Table 9d: 43 modified PCSK9 sense strands corresponding to the strands in Table 9a.

[0737]

[0738]

[0739]

[0740] The following Table 10a shows the nucleobase sequences of the antisense portion (e.g., the second nucleic acid portion) targeting PCSK9.

[0741]

[0742]

[0743] The following Table 10b shows the nucleobase sequences of the antisense portion (e.g., the second or fifth nucleic acid portion) targeting apolipoprotein C3 (APOC3).

[0744] Sequence number Experiment label Antisense sequence (5' to 3') 3422 AP277 UUGGAUAGGCAGGUGGACU 3423 AP337 UGCACUGAGAAUACUGUCC 3424 AP028 UCAACAAGGAGUACCCGGG 3425 AP369 UCUUGUCCAGCUUUAUUGG 3426 AP366 UUCCAGCUUUAUUGGGAGG 3427 AP367 UGUCCAGCUUUAUUGGGAG 3428 AP336 UCACUGAGAAUACUGUCCC

[0745] Table 11a below shows the nucleobase sequence of the sense part (third nucleic acid part) targeting PCSK9.

[0746] Sequence number Experimental label Sense strand sequence (5' to 3') 3429 PCS44 UUUGUAACUUGAAGA 3430 PCS46 CCUGUUUUGCUUUUA 3431 PCS53 UGUUUUGCUUUUGUA 3432 PCS29 UAGACCUGUUUUGCA 3433 PCS57 CUGUUUUGCUUUUGA 3434 PCS52 AAGAUAUUUAUUCUA 3435 PCS55 ACCUGUUUUGCUUUA

[0747] Table 11b below shows the nucleobase sequence of the sense part (fourth or sixth nucleic acid part) targeting apolipoprotein C3 (APOC3).

[0748] Sequence number Experimental label Sense strand sequence (5' to 3') 3436 AP277 CACCUGCCUAUCCAA 3437 AP337 AGUAUUCUCAGUGCA 3438 AP028 GGUACUCCUUGUUGA 3439 AP369 UAAAGCUGGACAAGA 3440 AP366 CCAAUAAAGCUGGAA 3441 AP367 CAAUAAAGCUGGACA 3442 AP336 CAGUAUUCUCAGUGA

[0749] Table 12a shows the antisense part targeting PCSK9 containing modification information.

[0750]

[0751] Table 12b shows the antisense part targeting apolipoprotein C3 (APOC3) containing modification information.

[0752]

[0753] Table 13a shows the sense part targeting PCSK9 containing modification information.

[0754]

[0755] Table 13b shows the sense part targeting apolipoprotein C3 (APOC3) containing modification information.

[0756]

[0757]

[0758] Table 14a shows the linked first and fourth nucleic acid parts of the (APOC3 + PCSK9) combination. These parts form a continuous single strand through direct connection.

[0759]

[0760]

[0761]

[0762]

[0763]

[0764] Table 14b shows the linked second and third nucleic acid parts of the (apolipoprotein C3 + proprotein convertase subtilisin / kexin type 9) combination. These parts are directly linked to form a continuous single strand.

[0765]

[0766]

[0767]

[0768]

[0769] The 5'-terminal nucleoside of the antisense (guide) strand (i.e., the first region as defined in the claims) can contain any nucleobase that can exist in an RNA molecule, that is, it can be any one of adenine (A), uracil (U), guanine (G), or cytosine (C). In addition, the 3'-terminal nucleoside of the sense (passenger) strand (i.e., the second region as defined herein) can also contain any nucleobase that can exist in an RNA molecule, that is, it can also be any one of adenine (A), uracil (U), guanine (G), or cytosine (C). However, advantageously, the nucleobase of the 3'-terminal residue should be complementary to the 5'-terminal nucleobase of the antisense (guide) strand (i.e., the first region as defined herein).

[0770] Although the methods shown and described herein are presented as a series of operations performed in a particular order, it should be understood that these methods are not limited by the order of operations. For example, the order in which certain operations occur may be different from that described herein. In addition, one operation can be performed simultaneously with another operation. Furthermore, in some cases, not all of the operations may be required to implement the methods described herein.

[0771] The order of steps of the methods described herein is for illustration only, and these steps can be performed in any suitable order and, where appropriate, simultaneously. In addition, steps can be added or replaced in any method, and individual steps can be deleted, without departing from the scope of the subject matter described herein. Aspects of any of the above examples can be combined with aspects of other examples to form more examples.

[0772] It should be understood that the above description of specific embodiments is for illustration only, and those skilled in the art can make various modifications. The above content includes examples of one or more embodiments. Of course, for the purpose of describing the above aspects, it is impossible to list every possible modification and variation of the above compounds, compositions, or methods, but those of ordinary skill in the art can recognize that there are many other modifications and changes possible for each aspect. Therefore, the aspects described are intended to cover all changes, modifications, and variations that fall within the scope of the appended claims. Specific Embodiments

[0773] The following examples illustrate certain embodiments of the present disclosure but do not constitute a limitation on the disclosure. Further, in providing specific embodiments, the inventors have considered the general application of these specific embodiments. For example, the disclosure of oligonucleotides having a specific motif or modification pattern provides reasonable support for other oligonucleotides having the same or similar motif or modification pattern.

[0774] The synthesis of the RNA interference (RNAi) constructs disclosed herein was performed using synthetic methods known to those skilled in the art, such as the synthetic methods disclosed at the website https: / / en.wikipedia.org / wiki / Oligonucleotide_synthesis (retrieved on February 16, 2022), the methods disclosed in this website are incorporated herein by reference in their entirety. The only difference from the synthetic methods disclosed in this reference is that N-acetylgalactosamine (GalNAc) phosphoramidite immobilized on a carrier was used in the first step of the synthetic method.

[0775] Example 1

[0776] Materials and Methods

[0777] Angiotensinogen (AGT) target identification and duplex preparation:

[0778] Oligomeric compounds targeting AGT were identified by bioinformatics analysis of the human AGT messenger ribonucleic acid (mRNA) sequence with the reference sequence number NM_000029.3. 100 compounds were selected and synthesized into mxRNA hairpin structures. These compounds were dissolved in molecular biology grade water to a concentration of 50 micromoles per liter (μM). The duplexes were annealed by heating at 95 degrees Celsius for 5 minutes and then gradually cooling to room temperature. The mxRNA was annealed by heating at 95 degrees Celsius for 5 minutes and then rapidly cooling on ice.

[0779] Cell culture:

[0780] Human primary hepatocytes (mixed samples from 5 donors - Sekisui Chemical Co., Ltd., product number HPCH05+) were thawed immediately before the experiment and cultured in 1-fold concentration complete Williams medium (Gibco, product number A1217601) supplemented with a hepatocyte seeding supplement kit (Gibco, product number CM3000). To ensure the stability of the compounds, the concentration of fetal bovine serum (FBS) was adjusted from 5% in the manufacturer's formulation to a final 2.5%.

[0781] AGT - Preliminary screening:

[0782] Human primary hepatocytes (mixed samples from 5 donors - Sekisui Chemical Co., Ltd., product number HPCH05+; 2 vials) were thawed by pouring the hepatocyte pellet into 45 ml of pre-warmed Sekisui OptiThaw hepatocyte medium (product number K8000), centrifuged at 250×g for 5 minutes, and then resuspended in 40 ml of 2-fold concentrated complete Williams medium (1-fold concentrated complete Williams medium contains: 2.5% FBS, 1 μmol / L dexamethasone, penicillin / streptomycin (100 units / ml / 100 μg / ml), 4 μg / ml human insulin, 2 mmol / L GlutaMAX, 15 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4) and counted. Then, the cells were seeded at a density of 25,000 cells per well in 50 μl of 2-fold concentrated complete Williams medium onto a 96-well type I rat tail collagen culture plate and allowed to settle and adhere for 5 hours before transfection. After settling, the compounds were further diluted to 2 μmol / L in basal Williams medium. 50 μl of each 2 μmol / L oligonucleotide compound targeting AGT (AGT_1 to AGT_100 in Table 3c) was added to the corresponding seeded hepatocytes to achieve a final concentration of 1 μmol / L in 100 μl of 1-fold concentrated complete Williams medium.

[0783] After 72 hours of transfection, according to the manufacturer's protocol, cells were harvested and RNA was isolated using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, product number 12173011A). The AGT expression level in the harvested RNA was detected by TaqMan real-time fluorescence quantitative polymerase chain reaction (qPCR) using the Luna Universal ProbeOne-Step RT-qPCR Kit (New England Biolabs, product number E3006). Multiplex qPCR was performed for each sample using the AGT TaqMan probe set (Hs01586213_m-FAM) and the universal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) VIC probe (Thermo Fisher Scientific, product number 4326317E). Thermal cycling and data acquisition were performed using the Applied Biosystems QuantStudio3 / 5 real-time PCR system.

[0784] Figure 1 Shows the results of the preliminary screening of selected oligomeric compounds targeting angiotensinogen (AGT) (compounds with sequence numbers 501 - 600 and 3604 - 3605 selected from Table 3c) and their activity in inhibiting AGT expression.

[0785] Example 2

[0786] Table 15 below shows the half maximal inhibitory concentration (IC 50 ) values (in nanomoles, nM) of 30 constructs (from Table 3c) selected according to the examples. These constructs were serially five-fold diluted in seven series starting from 1000 nM in basal Williams' medium (WEM). According to the protocol described in the above preliminary screening, the inhibitory effects of the constructs at different concentrations on angiotensinogen (AGT) messenger ribonucleic acid (mRNA) were determined. The maximum knockdown percentage (Max% KD) represents the maximum knockdown degree achievable at a concentration of 1000 nM, where 0% represents no knockdown and 100% represents complete knockdown. M4K4 was used as a reference. The inhibition percentages of the constructs at different concentrations were calculated, and the IC 50 values were determined using GraphPad Prism 9.0 software.

[0787] Table 15:

[0788]

[0789]

[0790] Figures 2a to 2h Shows the results of the inhibition of AGT gene expression after serial five-fold dilution of various oligonucleotide compounds targeting angiotensinogen (AGT) (from Table 3c).

[0791] The transmembrane serine protease 6 (TMPRSS6) construct used as a positive control has the following modified structure: 5'vP[mA][fA][mC][fC][mA][fG][mA][fA][mG][fA][mA][fG][mC][fA][mG][fG][mU][fG][iN][fC][mU][fG][fC][fU][mU][fC][mU][fU][mC][fU][mG][fG][mU][fU]#[3XGalNAc] (SEQ ID NO: 3587).

[0792] Example 3

[0793] Materials and Methods

[0794] Cell culture:

[0795] The human hepatocellular carcinoma cell line HepG2 (American Type Culture Collection number 85011430) was passaged every two weeks and cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.2), 1 mM sodium pyruvate, 1X minimum essential medium (MEM) non-essential amino acids, and 1X penicillin / streptomycin (i.e., complete EMEM medium).

[0796] Apolipoprotein C3 (APOC3) target identification and duplex preparation:

[0797] Targets targeting APOC3 were determined by bioinformatics analysis of the human APOC3 messenger ribonucleic acid (mRNA) sequence with the reference sequence number NM_000040. In particular, the construct should be able to target APOC3 mRNA regardless of its splice variants and isoforms. 376 targets were selected and synthesized into asymmetric duplexes (14 nucleotides for the sense strand and 19 nucleotides for the antisense strand). These compounds were dissolved in molecular biology grade water to a concentration of 50 micromoles per liter (μM), then heated at 95 degrees Celsius for 5 minutes, and then gradually cooled to room temperature for annealing. APOC3 - Preliminary screening:

[0798] On the day of transfection, HepG2 cells were collected by trypsin digestion, counted, and seeded at a density of 10,000 cells per well in 50 μL of complete EMEM medium containing 20% FBS in 96-well tissue culture-treated plates. After allowing the cells to settle for 4 hours, each APOC3 duplex was transfected into the cells in triplicate at a dose of 2 picomoles (pmoles) using RNAiMax transfection reagent (Thermo Fisher Scientific). Briefly, 8 picomoles of each duplex was diluted in 100 μL of OptiMEM medium and gently mixed with 0.8 μL of RNAiMax dissolved in 100 μL of OptiMEM medium to make a complex with a total volume of 200 μL. 50 μL of each RNAiMax complex duplex was added to the corresponding triplicate wells of HepG2 cells to give a final duplex concentration of 20 nanomoles (nM) in 100 μL of EMEM / OptiMEM medium containing 10% FBS and mixed 1:1.

[0799] After 72 hours of transfection, cells were harvested and RNA was isolated using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, product number 12173011A) according to the manufacturer's protocol. The expression level of APOC3 in the harvested RNA was detected by TaqMan real-time fluorescence quantitative polymerase chain reaction (qPCR) using the Luna Universal ProbeOne-Step RT-qPCR Kit (New England Biolabs, product number E3006). Two independent qPCR assays were performed for each sample using two different sets of APOC3 TaqMan probe sets and a universal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) VIC probe (Thermo Fisher Scientific, product number 4326317E). Thermal cycling and data acquisition were performed using an Applied Biosystems QuantStudio3 real-time PCR system. Based on the results of the primary screening, 77 oligomeric compounds were selected that could knockdown the target by at least 70% when evaluated using either set of probes. These 77 compounds were selected from the compounds with sequence numbers 601-1352 in Table 4a and Table 4b above. APOC3 - Secondary screening:

[0800] Based on the data from the primary screening, a more narrow range of dose-response curve tests were performed on the top 30 APOC3 duplexes. As before, HepG2 cells were harvested by trypsinization and seeded into 96-well tissue culture plates at a density of 10,000 cells per well in 50 μL of complete EMEM medium containing 20% FBS and allowed to adhere for 4 hours.

[0801] 36 picomoles of each duplex was dissolved in 180 μL of OptiMEM medium and gently mixed with 2.16 μL of RNAiMax dissolved in 180 μL of OptiMEM medium to form a transfection complex with a total volume of 360 μL. Then, it was serially diluted two-fold with basal OptiMEM medium. 50 μL of each dilution was added to the corresponding triplicate wells of HepG2 cells, resulting in a dilution series from 50 nM to 0.32 nM in 100 μL of EMEM / OptiMEM medium containing 10% FBS and mixed 1:1.

[0802] After 72 hours of transfection, cells were harvested and RNA was extracted using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, product number 12173011A) according to the manufacturer's protocol. The expression level of APOC3 in the extracted RNA was detected by TaqMan real-time fluorescence quantitative polymerase chain reaction (qPCR) using the Luna Universal ProbeOne-Step RT-qPCR Kit (New England Biolabs, product number E3006). Single qPCR assays were performed on each sample using the APOC3 TaqMan probe set and the universal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) VIC probe (Thermo Fisher Scientific, product number 4326317E). Thermal cycling and data acquisition were performed using the Applied Biosystems QuantStudio 3 Real-Time PCR System.

[0803] Example 4

[0804] Results

[0805] The following Table 16 shows the half-maximal inhibitory concentration (IC 50 ) values (in nanomoles per liter, nM) for 30 constructs selected according to the examples.

[0806]

[0807]

[0808] Half-maximal inhibitory concentration (IC 50 ) data in the single- to double-digit nanomolar range indicate that many of the constructs described herein perform excellently.

[0809] Example 5

[0810] Materials and methods

[0811] Cell culture:

[0812] Human primary hepatocytes (pooled samples from 5 donors - Jitsubishi Chemical Corporation, product number HPCH05+) were thawed immediately before the experiment and cultured in 1x complete Williams medium (Jitsubishi Chemical Corporation, product number A1217601) supplemented with the Hepatocyte Seeding Supplement Pack (Jitsubishi Chemical Corporation, product number CM3000). To ensure the stability of the compound, the concentration of fetal bovine serum (FBS) was adjusted from 5% in the manufacturer's formulation to 2.5% final.

[0813] The components of 1× concentration of complete Williams medium include: 2.5% FBS, 1 μmol / L dexamethasone, penicillin / streptomycin (100 U / mL / 100 μg / mL), 4 μg / mL human insulin, 2 mmol / L GlutaMAX, 15 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with a pH value of 7.4.

[0814] Hepatocytes were seeded on a 96-well tissue culture plate (Thermo Fisher Scientific, product number A1142803) coated with type I collagen (rat tail source).

[0815] Preparation of apolipoprotein C3 (APOC3) compounds:

[0816] The compound was dissolved in phosphate buffered saline (PBS) to a concentration of 10 mg / mL, then heated at 95 °C for 5 minutes, and subsequently rapidly cooled on ice for annealing.

[0817] Transfection of APOC3 compounds:

[0818] On the day of transfection, primary human hepatocytes were thawed in 45 mL of human OptiThaw medium (Wako Pure Chemical Industries, product number K8000) and centrifuged at 200×g for 5 minutes. The cells were resuspended in 2× concentration of complete Williams medium and counted. Then, the cells were seeded at a density of 25,000 cells per well in 50 μL of 2× concentration of complete Williams medium on a 96-well type I rat tail collagen culture plate, and allowed to settle and adhere for 4 hours before transfection.

[0819] The compound was further diluted to 2 μmol / L in basal Williams medium. A seven-step, five-fold serial dilution from 2 μmol / L to 0.000128 μmol / L was performed in basal Williams medium. 50 μL of each dilution was added to the corresponding triplicate seeded hepatocytes, such that the final dilution series in 100 μL of 1× concentration of complete Williams medium was from 1 μmol / L to 0.000064 μmol / L.

[0820] After 72 hours of transfection, cells were harvested and RNA was isolated using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, product number 12173011A) according to the manufacturer's protocol. The expression level of APOC3 in the harvested RNA was detected by TaqMan real-time fluorescence quantitative polymerase chain reaction (qPCR) using the Luna Universal ProbeOne-Step RT-qPCR Kit (New England Biolabs, product number E3006). Single qPCR assays were performed for each sample using the APOC3 TaqMan probe set (Hs00906501_g1-FAM) and the universal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) VIC probe (Thermo Fisher Scientific, product number 4326317E). Thermal cycling and data acquisition were performed using the Applied Biosystems QuantStudio 3 / 5 Real-Time PCR System.

[0821] Table 17: Constructs Used as Positive Controls

[0822]

[0823] Note: vP represents vinyl phosphonate; iN represents 2'-hydroxy inverted nucleotide

[0824] Experimental Results

[0825] From Figure 3a it can be seen that several variants of the A28 and A277 structures all exhibited excellent activity.

[0826] From Figure 3b it can be seen that all molecules demonstrated excellent activity. See also sequence numbers 2165 - 2188 in Table 6a - 6b.

[0827] Example 6

[0828] Research Protocol

[0829] The research protocol titled "Candidate Screening Study of mxRNA Lead Compounds in Male Human Liver-uPA-SCID Mice, Non-GLP Study" was drafted before the animal experiments and research were completed, so the future tense was used. However, since the research has been fully completed, in the following description of the research protocol, each "future tense" should be regarded as the "past tense". See Figure 4 .

[0830] Research Objectives

[0831] The purpose of this non-GLP study was to evaluate the dose and time-response effects of two selected mxRNA leads on a candidate N-acetylgalactosamine-small interfering RNA (GalNAc-siRNA) construct targeting apolipoprotein C3 (APOC3) using a human liver-uPA-SCID mouse model. The compounds will be administered by subcutaneous injection, and the mice will be sacrificed at 14 and 42 days for the two lead mxRNAs, respectively.

[0832] Before necropsy, plasma and serum will be collected. At necropsy, three liver tissue biopsy samples (2 mm) will be taken from each animal and stored separately in vials containing RNAlater, snap-frozen, and stored at -80°C. Another three liver tissue biopsy samples (2 mm) will be placed in the same vial, snap-frozen, and stored at -80°C.

[0833] Regulatory Compliance

[0834] This non-GLP study will not be conducted in accordance with the Good Laboratory Practice (GLP) regulations (21 CFR Part 58) of the US Food and Drug Administration (FDA).

[0835] Animal Welfare Compliance

[0836] The following described and implemented procedures will be carried out in accordance with the Guide for the Care and Use of Laboratory Animals, the Animal and Plant Health Inspection Service (USDA APHIS) of the United States Department of Agriculture, the Animal Welfare Act, and / or standard operating procedures.

[0837] This protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the testing facility.

[0838] Study Schedule

[0839] End date of acclimation / quarantine: ≥5 days

[0840] Date of baseline procedure: No baseline procedure

[0841] Date of start of procedure on Day 0: Tentative: December, pending test materials

[0842] Time of necropsy: Day 14 and Day 42 after treatment

[0843] Completion time of in-life study: 6 weeks after treatment

[0844] Preliminary report: Not required by the sponsor, only data will be provided

[0845] Final report release: Not required

[0846] Test System Information

[0847] Animal Testing

[0848] Common name: Mouse

[0849] Species / type: Rodent - human liver - uPA - SCID mouse

[0850] Number of animals (by sex): 36 males, all untreated, age range: 14 - 19 weeks

[0851] Weight range: approximately 20 grams

[0852] The mice used in this study are human liver - uPA - SCID mice, and approximately 80% of the hepatocytes in each mouse have been replaced by human hepatocytes. Those skilled in the art know the methods for preparing such mice, and at least some of the methods are described in the article published by P. Meuleman and G. Leroux - Roels in Antiviral Res. December 2008; 80(3):231 - 8, and the full text of this article is incorporated herein by reference.

[0853] Acclimation period

[0854] Duration:

[0855] All animals will undergo an acclimation period of at least 5 days before being approved for release by the attending veterinarian, at which time the overall health status of the animals will be evaluated. Animals that do not pass the acclimation period assessment will receive appropriate treatment and be further evaluated before release. All records of the acclimation period will be retained in the study file.

[0856] Method and location of animal identification:

[0857] Animals will be assigned consecutive numbers. Ear tags will be applied to the animals by punching holes or notching in the auricle under anesthesia to permanently identify each animal. Alternatively, tattoos can also be made on the animal's tail. A cage card will also be attached to each animal cage, indicating the animal number, sex, supplier, strain, principal investigator of the study, and study number.

[0858] Study design

[0859] Design details

[0860] This study will use one type of mouse, namely 36 human liver - uPA - SCID mice. The animals will be grouped according to treatment type, dose, and survival period. Each animal will receive the test material by subcutaneous injection. Control groups 1A and 1B each have 4 animals and receive phosphate - buffered saline (PBS) as a control dose. Experimental groups 2A, 2B, 2C, 3A, 3B, and 3C will receive a single dose (10 or 30 mg / kg) of treatment, and each dose group has 4 animals.

[0861] All animals will survive for 14 days or 42 days. For specific details, see Table 18 below.

[0862] Table 18: Research Grouping Table

[0863]

[0864] It is possible to collect more, with a minimum of 1.2 ml. This blood will be evenly distributed into serum separator tubes and plasma separator tubes. After separation (see Section 14.10), the serum will be evenly aliquoted into two separate vials, and the plasma will also be aliquoted into two separate vials, as shown below:

[0865] · 1.2 ml of blood = 0.6 ml for the serum separator tube and 0.6 ml for the plasma separator tube

[0866] · Serum (0.3 ml after separation) = 0.15 ml × 2 vials

[0867] · Plasma (0.3 ml after separation) = 0.15 ml × 2 vials

[0868] The above serum and plasma samples will be labeled, quickly frozen, and stored at -80°C.

[0869] If the blood collection volume exceeds the minimum of 1.2 ml, the excess blood will be placed in the serum separator tube. After processing, the serum will be transferred to a labeled vial and refrigerated at 4°C for rodent lipid analysis.

[0870] Note: Serum and plasma will be used for protein detection. During the operation, care should be taken to avoid hemolysis or coagulation.

[0871] During autopsy, three 2-mm biopsy tissue blocks will be taken from the left, middle, and right liver lobes respectively, placed in different vials, soaked in RNAlater (an RNA protectant) for 15 minutes, quickly frozen, and stored at -80°C. Additionally, three more 2-mm liver biopsy tissue blocks will be taken from the left, middle, and right liver lobes respectively and placed in the same vial, quickly frozen, and stored at -80°C. The remaining liver tissue will be quickly frozen and stored in a 10-ml conical tube at -80°C.

[0872] Research Design Changes

[0873] As the research progresses, this experimental protocol may be subject to changes. If the changes (to the protocol) are likely to have a negative impact on the research or the safety of the research subjects, approval from the Institutional Animal Care and Use Committee (IACUC) is required.

[0874] Animal Inclusion and Exclusion Criteria

[0875] Any animal determined to be unhealthy during veterinary pre-screening will be excluded from the study, and if there are alternative animals available, they will be used as replacements. For surviving animals that are found to be dead or near death after treatment, if there are alternative animals available, they can be replaced through a revision of the study protocol.

[0876] Animal Disposal

[0877] At the end of the study, the animals will be euthanized.

[0878] Route of Administration

[0879] Subcutaneous injection will be performed at the nape of the neck. The injection volume is 200 microliters.

[0880] Results

[0881] Figure 5 Highlighted is the dose-response effect of the percentage reduction of APOC3 mRNA in liver tissue and the level of APOC3 protein in plasma in animals treated with different mxRNA constructs (A28(14-4)mF, SEQ ID NOs: 2176, 2188, and A277(12-5), SEQ ID NOs: 2169, 2181) compared to control animals on day 14.

[0882] In addition, regarding Figure 5 the following explanations are provided:

[0883] A28(14-4)mF-10 = A28(14-4)mF 10 mg / kg dose group

[0884] A28(14-4)mF-30 = A28(14-4)mF 30 mg / kg dose group

[0885] A277(12-5)-10 = A277(12-5) 10 mg / kg dose group

[0886] A277(12-5)-30 = A277(12-5) 30 mg / kg dose group

[0887] Figure 6 Highlighted is the dose-response effect of the average percentage reduction of triglycerides and total cholesterol in serum in animals treated with different mxRNA constructs targeting APOC-3 (A28(14-4)mF, SEQ ID NOs: 2176, 2188, and A277(12-5), SEQ ID NOs: 2169, 2181) at 10 mg / kg and 30 mg / kg doses compared to control animals on day 14.

[0888] Figure 7a and 7bHighlights the duration effect of the mean percent reduction of APOC3 mRNA in liver tissue and the plasma APOC3 protein level in animals treated with different APOC3-targeting mxRNA (10 mg / kg) constructs (A28(14-4)mF, SEQ ID NOs: 2176, 2188, and A277(12-5), SEQ ID NOs: 2169, 2181) at day 14 (week 2) and week 6 compared to control animals. Also note regarding these figures that one outlier was excluded from the A277(12-5) group.

[0889] Figure 8a and 8b Highlights the duration effect of the mean percent reduction of triglycerides (TG) and total cholesterol (TC) in serum in animals treated with different APOC3-targeting mxRNA (10 mg / kg) constructs (A28(14-4)mF, SEQ ID NOs: 2176, 2188, and A277(12-5), SEQ ID NOs: 2169, 2181) at day 14 (week 2) and week 6 compared to control animals. Note regarding these figures that one outlier was excluded from the A277(12-5) group.

[0890] A28(14-4)mF APOC3-targeting mxRNA construct (SEQ ID NOs: 2176, 2188)

[0891] · The APOC3 mRNA inhibition rate reached 88% at week 2 compared to the control group and was maintained at 78% at week 6.

[0892] · The plasma APOC3 level decreased by 90% at week 6 compared to the control group and remained at 85% at week 6.

[0893] · The serum triglyceride level decreased by 32% at week 2 compared to the control group and the decrease increased to 41% at week 6.

[0894] · The serum total cholesterol level decreased by 43% at week 2 compared to the control group and was maintained at 33% at week 6. A277(12-5) APOC3-targeting mxRNA construct (SEQ ID NOs: 2169, 2181)

[0895] · The APOC3 mRNA inhibition rate reached 56% at week 2 compared to the control group and was maintained at 42% at week 6.

[0896] · The plasma APOC3 level decreased by 83% at week 6 compared to the control group and remained at 84% at week 6.

[0897] · Compared with the control group, the serum triglyceride level decreased by 8% at week 2 and the decrease increased to 52% at week 6.

[0898] · Compared with the control group, the serum total cholesterol level decreased by 36% at week 2, but this effect disappeared at week 6.

[0899] Conclusion

[0900] The A28(14-4)mF construct showed excellent activity, with a 98% downregulation rate of the target protein at week 2 at a dose of 30 mg / kg. In addition, the A28(14-4)mF construct maintained excellent (protein knockdown) activity at both week 2 and week 6 at a dose of 10 mg / kg.

[0901] Example 7

[0902] According to the protocol described in detail in Example 6, the compound A28(14-4)mF (also known as STP125G) was observed for its effects over a longer period. An overview of this extended study is shown in Figure 9 .

[0903] The corresponding results are shown in Figure 10 (APOC3 mRNA and protein knockdown) and Figure 11 (triglyceride and total cholesterol).

[0904] There are several aspects worthy of note:

[0905] · A single dose of 10 mg / kg was sufficient to achieve mRNA and protein knockdown over a six-week period, but a rebound phenomenon gradually appeared towards the end of the study.

[0906] · Not only was the triglyceride, a blood lipid component mainly related to APOC3, downregulated, but surprisingly, the total cholesterol was also downregulated.

[0907] · When evaluating this latter finding, the characteristics of the mice used in this study must be fully considered. As Figure 12 shown, it is estimated that 20%-25% of the cells in the humanized liver are still murine cells. A28(14-4)mF does not target murine APOC3. Therefore, the unsilenced murine APOC3 affects the observed triglyceride and total cholesterol levels. In other words, in a (pure) human system, it is expected that the downregulation of these two blood lipids will exceed the observations in this study.

[0908] Example 8

[0909] Materials and Methods

[0910] Cell Culture

[0911] HepG2 cells (American Type Culture Collection No. 85011430) were passaged every two weeks and cultured in Eagle's Minimum Essential Medium (EMEM, complete medium) supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.2), 1 mM sodium pyruvate, 1x concentration of MEM non-essential amino acids, and 1x concentration of penicillin / streptomycin (Pen / Strep).

[0912] PCSK9 Target Identification and Double-Strand Preparation

[0913] The target of PCSK9 was determined by bioinformatics analysis of the human PCSK9 mRNA sequence (reference sequence NM_174936.3). 250 targets were selected and asymmetric double-strands (15 sense strands, 19 antisense strands) were synthesized. The compound was dissolved in molecular biology-grade water to prepare a 50 μM solution, heated at 95 °C for 5 minutes, and then gradually cooled to room temperature for annealing treatment.

[0914] PCSK9 - Primary Screening

[0915] On the day of transfection, HepG2 cells were collected by trypsin digestion, counted, and seeded into 96-well tissue culture-treated plates at a density of 10,000 cells per well, with 50 μL of complete EMEM medium containing 20% FBS added to each well. After the cells were allowed to stand for 4 hours, each PCSK9 double-strand (2 picomoles) was transfected into the cells in triplicate using RNAiMax (Thermo Fisher Scientific). Briefly, 8 picomoles of each double-strand was diluted in 100 μL of OptiMEM medium, then gently mixed with 0.8 μL of RNAiMax in 100 μL of OptiMEM medium to form a complex with a total volume of 200 μL. 50 μL of each RNAiMax complex double-strand was added to the corresponding triplicate wells of HepG2 cells, resulting in a final mixture with a volume of 100 μL, a final double-strand concentration of 20 nM, an EMEM / OptiMEM ratio of 50 / 50, and containing 10% FBS.

[0916] After 72 hours of transfection, cells were harvested and RNA was extracted using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, 12173011A) according to the manufacturer's protocol. The extracted RNA was assayed for PCSK9 expression by Taqman quantitative polymerase chain reaction (qPCR) using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006). Multiplex assays were performed on each sample using two different sets of PCSK9 Taqman probe sets (Hs00545399_m1-FAM and Hs03037355_m1-FAM) and the universal GAPDH VIC probe (Thermo Fisher Scientific, 4326317E), and two independent qPCR assays were carried out. Thermal cycling and data acquisition were performed using an Applied Biosystems QuantStudio 3 Real-Time PCR System.

[0917] PCSK9 - Secondary Screening

[0918] Based on the primary screening data, dose-response curves were tested for the 27 best-performing PCSK9 duplexes, and the half-maximal inhibitory concentration (IC50) values were determined. As before, HepG2 cells were harvested by trypsin digestion and seeded in 96-well tissue culture plates at a density of 10,000 cells per well in 50 μL of complete EMEM medium containing 20% FBS, and the cells were allowed to settle for 4 hours. Each 36 picomoles of each duplex was gently mixed with 2.16 μL of RNAiMax in 180 μL of OptiMEM medium to make a transfection complex with a total volume of 360 μL. Then, a two-fold dilution series was made with basal OptiMEM medium. Fifty microliters of each dilution was added to the corresponding triplicate wells of HepG2 cells, resulting in a final volume of 100 μL, a dilution series from 50 nM to 0.32 nM, an EMEM / OptiMEM ratio of 50 / 50, and a mixture containing 10% FBS.

[0919] After 72 hours of transfection, cells were harvested and RNA was extracted using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, 12173011A) according to the manufacturer's protocol. The extracted RNA was used to detect the expression of PCSK9 by Taqman qPCR, using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006). Multiplex detection was performed for each sample using the PCSK9 Taqman probe set Hs00545399_m1-FAM and the universal GAPDH VIC probe (Thermo Fisher Scientific, 4326317E), and one qPCR assay was carried out. Thermal cycling and data acquisition were performed using the Applied Biosystems QuantStudio 3 Real-Time PCR System.

[0920] Example 9

[0921] Results obtained by performing the method of Example 8

[0922] Figure 13 Showed the results of the primary screening (knockdown percentage).

[0923]

[0924]

[0925] Example 10

[0926] Optimized double-stranded constructs

[0927] The complete definitions of these constructs are given in Table 20 below. AS represents the antisense strand (also referred to herein as the first region); SS represents the sense strand (also referred to herein as the second region). Further explanations of the relevant notations are provided above.

[0928] Table 20

[0929]

[0930] Performance data are shown in Figure 15 and Figure 16 . Comparison with inclisiran-like molecules ( Figure 16 ) showed that these constructs had excellent performance. In fact, the performance of two of the constructs was very similar to that of the trisialylated mannosamine (3xGalNAc, "toothbrush" type) derivative of inclisiran.

[0931] Example 11

[0932] Optimized hairpin-shaped molecules (mxRNA)

[0933] The complete definitions of these molecules are given in Table 21 below.

[0934]

[0935]

[0936] Performance data can be found in Figure 17 。

[0937] Example 12

[0938] Materials and Methods

[0939] Cell culture:

[0940] HepG2 cells (American Type Culture Collection No. 85011430) were passaged every two weeks and cultured in Eagle's Minimum Essential Medium (EMEM, complete medium) supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.2), 1 mM sodium pyruvate, 1x concentration of MEM non-essential amino acids, and 1x concentration of penicillin / streptomycin (Pen / Strep).

[0941] Screening of PCSK9 and APOC3 combinations and compound preparation:

[0942] The best-performing PCSK9 and APOC3 sequences were selected from Tables 14a - 14b above, and 49 combinatorial compounds were designed and synthesized as candidates, as shown in Tables 22a and 22b below. These compounds were dissolved in molecular biology-grade water to prepare a 50 μM solution, heated at 95 °C for 5 minutes, and then gradually cooled to room temperature for annealing treatment.

[0943] APOC3 / PCSK9 combination - screening

[0944] Based on the screening data of the PCSK9 and APOC3 duplexes, the best-performing APOC3 and PCSK9 sequences were combined into 49 candidates, and dose-response curve tests were conducted. HepG2 cells were collected by trypsin digestion and seeded in 96-well tissue culture plates at a density of 10,000 cells per well. 50 μL of complete EMEM medium containing 20% FBS was added to each well, and the cells were allowed to stand for 4 hours. 36 picomoles of each duplex was gently mixed with 2.16 μL of RNAiMax in 180 μL of OptiMEM medium to make a transfection complex with a total volume of 360 μL. Then, a two-fold dilution series was made with basal OptiMEM medium. 50 μL of each dilution was added to the corresponding triplicate wells of HepG2 cells, resulting in a final mixture with a volume of 100 μL, a dilution series from 50 nM to 0.32 nM, an EMEM / OptiMEM ratio of 50 / 50, and containing 10% FBS.

[0945] After 72 hours of transfection, cells were harvested and RNA was extracted using the PureLink Pro 96 Total RNA Purification Kit (Thermo Fisher Scientific, 12173011A) according to the manufacturer's protocol. The expression of PCSK9 and APOC3 in the extracted RNA was detected by Taqman qPCR using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006). For each sample, multiplex detection was performed using either the APOC3 Taqman probe (Hs00906501_g1-FAM) or the PCSK9 probe (Hs00906501_g1-FAM) and the universal GAPDH VIC probe (Thermo Fisher Scientific, 4326317E), with one qPCR detection for each. Thermal cycling and data acquisition were performed using an Applied Biosystems QuantStudio 3 real-time PCR system.

[0946] Example 13

[0947] Results

[0948] The following Tables 22a and 22b show the half-maximal inhibitory concentration (IC 50 ) values (in nanomoles, nM) for the specific constructs selected according to the examples. Table 22a shows the knockdown of PCSK9, and Table 22b shows the knockdown of APOC3. The numbers after "SR1-" in the first column correspond to the construct numbers in Tables 14a and 14b above.

[0949] Table 22a:

[0950]

[0951]

[0952] Table 22b:

[0953]

[0954]

[0955] The half maximal inhibitory concentration (IC 50 ) data is in the single-digit to double-digit nanomolar range, indicating excellent performance of numerous constructs. Notably, any one of the two target sites in the dual-target constructs can be knocked down by multiple constructs.

[0956] Example 14

[0957] Evaluation of the targeted human angiotensinogen (mxRNA) candidate in a humanized liver-uPA-SCID mouse (PXB) model Dose-response study of the lead compound, non-GLP study

[0958] Materials and Methods

[0959] 1. Research Objectives

[0960] This non-GLP (non-Good Laboratory Practice) study aims to evaluate the dose response of N-acetylgalactosamine (GalNAc)-conjugated mxRNA constructs targeting human angiotensinogen (AGT) in humanized liver-uPA-SCID (PXB) mice. The compound will be administered by subcutaneous injection, and the mice will survive for up to 14 days.

[0961] 2. Test System Information

[0962] 2.1 Animal Experiments

[0963] 2.1.1 Generic Name: Mouse

[0964] 2.1.2 Breed / Type: Rodent - Mouse PXB

[0965] 2.1.3 Number of Animals (by Gender): 32 male PXB mice, all untreated 2.1.4 Age Range: PXB mice at 14 - 19 weeks of age

[0966] 2.1.5 Weight Range: All mice are approximately 20 grams

[0967] 2.2 Adaptation Period

[0968] 2.2.1 Duration:

[0969] All animals will have an adaptation period of at least five (5) days before being released by the attending veterinarian, at which time the overall health status of the animals will be evaluated.

[0970] 3. Research Design

[0971] This study will use a type of mouse, namely 32 PXB mice. The animals will be grouped according to treatment type, dose, and survival period. Each animal will be treated by subcutaneous injection of the test substance.

[0972] · Group 1A has 4 animals and receives the PBS control dose.

[0973] · Groups 2A, 2B, and 2C have 4 / 5 animals each and receive single doses of 5 mg / kg, 10 mg / kg, and 30 mg / kg of (AGT_27), respectively.

[0974] · Groups 3A, 3B, and 3C have 4 / 5 animals each and receive single doses of 5 mg / kg, 10 mg / kg, and 30 mg / kg of (AGT_52), respectively.

[0975] The animals will survive for 14 days. See Study Table 23 for details.

[0976] At autopsy, three 2-mm biopsy tissue blocks will be taken from the left, middle, and right liver lobes and placed in different vials, soaked in RNAlater (an RNA protectant) for 15 minutes, and stored at -80 °C after rapid freezing. Additionally, three more 2-mm liver biopsy tissue blocks will be taken from the left, middle, and right liver lobes and placed in the same vial, stored at -80 °C after rapid freezing. The remaining liver tissue will be rapidly frozen and stored in a 10-ml conical tube at -80 °C.

[0977] Table 23: Study Table

[0978]

[0979]

[0980] 3.1 Test Drug 1

[0981] 3.1.1 Identification: AGT_27

[0982] 3.1.2 Manufacturer: Sirnaomics

[0983] 3.1.3 Description: N-acetylgalactosamine (GalNAc)-modified mxRNA targeting human angiotensinogen (AGT) mRNA

[0984] 3.1.4 Batch number: Will be recorded on the study materials form.

[0985] 3.1.5 Expiration date: Will be recorded on the study materials form.

[0986] 3.1.6 Storage temperature: 4 °C

[0987] 3.1.7 Biological hazard status: None

[0988] 3.1.8 Material Safety Data Sheet (MSDS): To be determined

[0989] 3.1.9 Appearance: Clear liquid

[0990] 3.1.10 Dosage information: See Table 1

[0991] 3.1.11 Storage of remaining test articles: None

[0992] 3.2 Test drug 2

[0993] 3.2.1 Identification: AGT_52

[0994] 3.2.2 Manufacturer: Sirnaomics

[0995] 3.2.3 Description: N-acetylgalactosamine (GalNAc)-modified mxRNA targeting human angiotensinogen (AGT) mRNA

[0996] 3.2.4 Batch number: To be recorded on the research material form

[0997] 3.2.5 Expiry date: To be recorded on the research material form

[0998] 3.2.6 Storage temperature: 4°C

[0999] 3.2.7 Biological hazard status: None

[1000] 3.2.8 Material Safety Data Sheet (MSDS): To be determined

[1001] 3.2.9 Appearance: Clear liquid

[1002] 3.2.10 Dosage information: See Table 1

[1003] 3.2.11 Storage of remaining test articles: None

[1004] For the schematic diagram of this study, please refer to Figure 18 .

[1005] Results

[1006] The results are shown in Figure 19. Specifically, Figure 19 shows that both of the two molecules tested can significantly and dose-dependently reduce the mRNA and protein levels. Given that AGT-27 performs better, this molecule will be further studied in the following examples.

[1007] Example 15

[1008] Evaluation of the sustained effect of the targeted human angiotensinogen (AGT) mxRNA in a humanized liver-uPA-SCID mouse (PXB) model Non-GLP study

[1009] Materials and Methods

[1010] 1. Research Objectives

[1011] This non-GLP (Good Laboratory Practice) study was designed to evaluate the sustained effects of mxRNA targeting human angiotensinogen (AGT) - AGT-27A (SEQ ID NO: 3604) in humanized liver-uPA-SCID (PXB) mice. The compound will be administered by subcutaneous injection, and the mice will be maintained for up to 84 days.

[1012] 2. Test System Information

[1013] 2.1 Animal Experiments

[1014] 2.1.1 Generic Name: Mouse

[1015] 2.1.2 Breed / Type: Rodent - Mouse PXB

[1016] 2.1.3 Number of Animals (by Gender): 40 male PXB mice, all untreated

[1017] 2.1.4 Age Range: PXB mice at 14 - 19 weeks of age

[1018] 2.1.5 Weight Range: All mice approximately 20 g

[1019] 2.2 Adaptation Period

[1020] 2.2.1 Duration: All animals will undergo an adaptation period of at least seven (7) days before being released by the attending veterinarian, at which time the overall health of the animals will be evaluated.

[1021] 3. Study Design

[1022] 3.1 Design Details

[1023] This study will use one type of mouse, namely 40 PXB mice. The animals will be grouped according to treatment type, dose, and survival period. Each animal will be treated by subcutaneous injection of the test substance.

[1024] ● Groups 1A, 1B, 1C, and 1D: Each group has 5 animals and receives a single dose of PBS as a control.

[1025] ● Groups 2A, 2B, 2C, and 2D: Each group has 5 animals and receives a single dose of 30 mg / kg of AGT-27A.

[1026] The animals will be maintained for 14 days, 28 days, 56 days, and 84 days respectively. Details are shown in Table 24.

[1027] Before autopsy, the animals will be deeply anesthetized and terminal blood samples will be collected via the vena cava. The blood will be collected in plasma separation tubes.

[1028] Note: Plasma will be used for protein detection. Care should be taken to avoid hemolysis or coagulation.

[1029] During autopsy, three 2-mm biopsy tissue blocks will be taken from each of the left, middle, and right hepatic lobes and placed separately in different vials. They will be soaked in RNAlater for 15 minutes, quickly frozen, and stored at -80°C. Additionally, three more 2-mm liver biopsy tissue blocks will be taken from each of the left, middle, and right hepatic lobes and placed in the same vial, quickly frozen, and stored at -80°C. The remaining liver tissue will be quickly frozen, stored in a 10-ml conical tube, and kept at -80°C.

[1030] Table 24: Research Table

[1031]

[1032]

[1033] 3.2 Test article 1

[1034] 3.2.1 Identification: AGT-27A

[1035] 3.2.2 Manufacturer: Sirnaomics

[1036] 3.2.3 Description: GalNAc-modified mxRNA targeting human angiotensinogen (AGT) messenger ribonucleic acid (mRNA)

[1037] 3.2.4 Batch number: Will be recorded on the research materials form.

[1038] 3.2.5 Expiration date: Will be recorded on the research materials form.

[1039] 3.2.6 Storage temperature: 4°C

[1040] 3.2.7 Biological hazard status: None

[1041] 3.2.8 Safety Data Sheet (SDS): To be determined

[1042] 3.2.9 Appearance: Clear liquid

[1043] 3.2.10 Dosage information: See Table 1

[1044] 3.2.11 Storage of remaining test article: None

[1045] For a schematic diagram of this study, please also refer to Figure 20 .

[1046] Result

[1047] The results are shown in Figure 21. Apparently, a single administration produced a lasting effect on both the mRNA levels in the liver and the protein levels in the plasma.

Claims

1. An oligomeric compound capable of inhibiting the expression of angiotensinogen (AGT), which comprises at least one first region composed of linked nucleosides, the first region having at least one first nucleobase sequence that is at least partially complementary to at least a part of the RNA transcribed from the AGT gene, wherein the first nucleobase sequence is selected from the sequences in Table 1a (SEQ ID NOs: 1 to 100) or a partial sequence thereof, and the partial sequence optionally has a length of at least 18 nucleosides.

2. The oligomeric compound according to claim 1, further comprising at least one second region composed of linked nucleosides, the second region having at least one second nucleobase sequence that is at least partially complementary to the first nucleobase sequence and is selected from the sequences in Table 1b (SEQ ID NOs: 101 to 200) or a partial sequence thereof, and the partial sequence optionally has a length of at least 8, 9, 10 or 11 nucleosides.

3. The oligomeric compound according to claim 1 or 2, wherein the first nucleobase sequence is selected from the following sequences or a partial sequence thereof: SEQ ID NOs: 27, 44, 41, 97, 90, 62, 52, 93, 49, 73, 18, 37, 56, 100, 40, 75, 30, 42, 81, 17, 34, 53, 29, 26, 74, 94, 14, 3, 7 and 2.

4. The oligomeric compound according to claim 3, wherein the second nucleobase sequence is selected from the following sequences or a partial sequence thereof: SEQ ID NOs: 127, 144, 141, 197, 190, 162, 152, 193, 149, 173, 118, 137, 156, 200, 140, 175, 130, 142, 181, 117, 134, 153, 129, 126, 174, 194, 114, 103, 107 and 102.

5. The oligomeric compound according to any one of claims 1 to 4, wherein the first nucleobase sequence is selected from the following sequences or a partial sequence thereof: SEQ ID NOs: 27, 52, 56, 62, 75 and 93.

6. The oligomeric compound according to claim 5, wherein the second nucleobase sequence is selected from the following sequences or a partial sequence thereof: SEQ ID NOs: 127, 152, 156, 162, 175 and 193.

7. The oligomeric compound according to any one of claims 1 to 6, wherein the first region composed of linked nucleosides consists essentially of 18 to 35, 18 to 20, 18 or 19, or 19 linked nucleosides.

8. The oligomeric compound according to any one of claims 2 to 7, wherein the second region composed of linked nucleosides consists essentially of 10 to 35, 10 to 20, 10 to 16, 10 to 15, or 13, 14 or 15 linked nucleosides.

9. The oligomeric compound according to any one of claims 2 to 8, comprising at least one complementary double-stranded region, the double-stranded region comprising at least a portion of the first region consisting of linked nucleosides, the first region being directly or indirectly linked to at least a portion of the second region consisting of linked nucleosides, wherein optionally, the length of the double-stranded region is 10 to 19, 12 to 19, 12 to 15, or 14 or 15 base pairs, and wherein optionally, there is one mismatch within the double-stranded region.

10. The oligomeric compound according to claim 9, wherein both the first region and the second region consisting of linked nucleosides have a 5′ to 3′ directionality, thereby defining their 5′ and 3′ regions, respectively.

11. The oligomeric compound according to claim 10, wherein the 5′ region of the first region consisting of linked nucleosides is directly or indirectly linked to the 3′ region of the second region consisting of linked nucleosides, optionally by complementary base pairing, and wherein optionally, the 5′-terminal nucleoside of the first nucleoside region base pairs with the 3′-terminal nucleoside of the second nucleoside region.

12. The oligomeric compound according to claim 10 or 11, wherein the 3′ region of the first region of linked nucleosides is directly or indirectly linked to the 5′ region of the second region of linked nucleosides, wherein optionally, the first nucleoside region is directly and covalently linked to the second nucleoside region by means such as a phosphate ester, a phosphorothioate, or a dithiophosphate, and wherein optionally, the 3′-terminal nucleoside of the first region of linked nucleosides is directly and covalently linked to the 5′-terminal nucleoside of the second region of linked nucleosides by a phosphate ester, a phosphorothioate, or a dithiophosphate.

13. The oligomeric compound according to any one of claims 1 to 12, further comprising one or more ligands.

14. The oligomeric compound according to claim 13, wherein the one or more ligands, particularly two or more or three ligands, are conjugated to the second region of the linked nucleosides and / or the first region of the linked nucleosides.

15. The oligomeric compound according to claim 14, which depends from claim 10, wherein the one or more ligands are conjugated in the 3′ region, optionally at the 3′-terminal nucleoside of the second region and / or the first region of the linked nucleosides, and / or are conjugated to the 5′-terminal nucleoside of the second region of the linked nucleosides.

16. The oligomeric compound according to any one of claims 13 to 15, wherein the one or more ligands are any cell-targeting moieties, such as lipids, carbohydrates, aptamers, vitamins, and / or peptides that bind to specific targets on the cell membrane or cell surface.

17. The oligomeric compound according to claim 16, wherein the one or more ligands comprise one or more carbohydrates.

18. The oligomeric compound according to claim 17, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

19. The oligomeric compound according to claim 18, wherein the one or more carbohydrates comprise or consist of one or more hexose moieties.

20. The oligomeric compound according to claim 19, wherein the one or more hexose moieties are one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

21. The oligomeric compound according to claim 20, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

22. The oligomeric compound according to claim 21, comprising two or more N-acetylgalactosamine moieties, optionally three.

23. The oligomeric compound according to any one of claims 13 to 22, wherein the one or more ligands are attached to the oligomeric compound in a linear configuration or a branched configuration, optionally attached to a second region of the linked nucleoside.

24. The oligomeric compound according to claim 23, wherein the one or more ligands are attached to the oligomeric compound in a bi-antennary or tri-antennary configuration.

25. The oligomeric compound according to any one of claims 1 to 24, wherein the compound consists of a first region of the linked nucleoside and a second region of the linked nucleoside.

26. The oligomeric compound according to any one of claims 1 to 24, wherein there is an intervening third region of the linked nucleoside between the first region and the second region.

27. The oligomeric compound according to claim 26, wherein the oligomeric compound comprises or consists of a single strand having the first, third, and second nucleoside regions or consisting of the same, wherein at least a portion of the first nucleoside region is directly or indirectly connected to at least a portion of the second nucleoside region, thereby forming the at least partially complementary double-stranded region.

28. The oligomeric compound according to any one of claims 9 to 25, wherein the oligomeric compound comprises or consists of a single strand having the first and second regions of the linked nucleoside or consisting of the same, wherein at least a portion of the first region of the linked nucleoside is directly or indirectly connected to at least a portion of the second region of the linked nucleoside, thereby forming the at least partially complementary double-stranded region.

29. The oligomeric compound according to claim 28, wherein the first and second nucleoside regions are directly adjacent on the single strand.

30. The oligomeric compound according to claim 28 or 29, wherein the first nucleoside region has a greater number of linked nucleosides than the second nucleoside region, wherein optionally the ratio of the total number of linked nucleosides in the first nucleoside region to the total number of linked nucleosides in the second nucleoside region is from about 19 / 15 to about 19 / 8, or from about 18 / 15 to about 18 / 8; and / or the percentage of the total number of linked nucleosides in the first nucleoside region relative to the total number of nucleosides in the oligomeric compound is from about 55% to about 60%.

31. The oligomeric compound according to claim 30, wherein additional linked nucleosides in the first nucleoside region form a hairpin loop connecting the first and second regions of the linked nucleosides, wherein optionally, a part of the first nucleobase sequence complementary to the RNA transcribed from the AGT gene forms the hairpin loop, and wherein the loop contains 2 to 5, or 4 or 5 nucleosides.

32. The oligomeric compound according to any one of the preceding claims, wherein the single strand is selected from Table 2, in particular from the group consisting of SEQ ID NOs: 227, 252, 256, 262, 275, and 293, and wherein optionally, the nucleobase of the 5'-terminal nucleoside of the first region of the linked nucleosides is replaced by uracil (U), and the nucleobase of the 5'-terminal nucleoside of the second region of the linked nucleosides is replaced by adenine (A).

33. The oligomeric compound according to claim 32, wherein the single strand is selected from the group consisting of SEQ ID NOs: 527, 552, 556, 562, 575, 593, 3604, and 3605, and wherein optionally, the nucleobase of the 5'-terminal nucleoside of the first region of the linked nucleosides is replaced by uracil (U), and the nucleobase of the 5'-terminal nucleoside of the second region of the linked nucleosides is replaced by adenine (A).

34. The oligomeric compound according to claim 33, which depends on claim 10, wherein the hairpin loop is located at the 3'-terminal region of the first region of the linked nucleosides, where, When the nucleobases of one, two, or more 3'-terminal nucleosides of the first nucleobase sequence permit, optionally, these 3'-terminal nucleosides fold back and form or contribute to the second region of the linked nucleosides.

35. The oligomeric compound according to claim 26 or 27, wherein the third nucleoside region, and optionally the 3'-terminal portion of the first nucleoside region (optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides) and / or the 5'-terminal portion of the second nucleoside region (optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides) form a hairpin loop.

36. The oligomeric compound according to any one of claims 29 to 31, wherein the hairpin loop contains 1 to 8, 2 to 7, 3 to 6, or 4 or 5 linked nucleosides.

37. The oligomeric compound according to any one of claims 1 to 36, which contains internucleoside linkage bonds, and wherein at least one internucleoside linkage bond is a modified internucleoside linkage bond.

38. The oligomeric compound according to claim 37, wherein the modified internucleoside linkage bond is a phosphorothioate or dithiophosphate internucleoside linkage bond.

39. The oligomeric compound according to claim 38, which contains 1 to 16 phosphorothioate or dithiophosphate internucleoside linkage bonds.

40. The oligomeric compound according to claim 39, which contains 7, 8, 9, or 10 phosphorothioate or dithiophosphate internucleoside linkage bonds.

41. The oligomeric compound according to any one of claims 38 to 40, which depends on claim 10, and which contains one or more phosphorothioate or dithiophosphate internucleoside linkage bonds in the 5'-terminal region of the first region of the linked nucleosides.

42. The oligomeric compound according to any one of claims 38 to 41, which depends on claim 10, comprising one or more phosphorothioate or phosphorodithioate internucleoside linkages in the 5′-terminal region of the second region of the linked nucleosides, wherein, Optionally, the oligomeric compound contains three phosphorothioate internucleoside linkage bonds at three adjacent nucleosides in the 5'-terminal region.

43. An oligomeric compound according to any one of claims 38 to 42, which depends on any one of claims 30 to 32, comprising phosphorothioate or phosphorodithioate internucleoside linkages between at least two, at least three, at least four or at least five adjacent nucleosides in the hairpin loop, according to the number of nucleosides present in the hairpin loop.

44. The oligomeric compound according to claim 43, comprising phosphorothioate or phosphorodithioate internucleoside linkages between each adjacent nucleoside present in the hairpin loop.

45. The oligomeric compound according to any one of claims 1 to 44, wherein at least one nucleoside comprises a modified sugar.

46. The oligomeric compound according to claim 45, wherein the modified sugar is selected from 2'-modified sugars, conformationally restricted nucleoside (CRN) sugars (such as locked nucleic acid (LNA) sugars, (S)-constrained ethyl bicyclic nucleic acid, constrained ethyl (cEt) sugars), tricyclic DNA, morpholino, unlocked nucleic acid (UNA) sugars, glycerol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA).

47. The oligomeric compound according to claim 46, wherein the 2′-modified sugar is selected from 2'-O-alkyl modified sugars, 2′-O-methyl modified sugars, 2'-O-methoxyethyl modified sugars, 2′-O-allyl modified sugars, 2'-C-allyl modified sugars, 2′-deoxy modified sugars (such as 2'-deoxyribose), 2′-F modified sugars, 2'-arabino-fluoro modified sugars, 2'-O-benzyl modified sugars, and 2'-O-methyl-4-pyridyl modified sugars.

48. The oligomeric compound according to claim 47, wherein at least one modified sugar is a 2'-O-methyl modified sugar.

49. The oligomeric compound according to claim 47 or 48, wherein at least one modified sugar is a 2'-F modified sugar, and optionally, at most 16 or 17 sugars are 2'-F modified sugars.

50. The oligomeric compound according to claim 48 or 49, wherein the sugar is ribose.

51. The oligomeric compound according to any one of claims 48 to 50, which depends on claim 10, wherein the sugar of the nucleoside at any position downstream of the first nucleoside at the 5′-end region of the first region of the linked nucleosides at the 2nd and 14th positions does not comprise a 2'-O-methyl modification.

52. The oligomeric compound according to any one of claims 48 to 51, wherein the 3′-end position of the second region of the linked nucleosides does not comprise a 2'-O-methyl modification.

53. The oligomeric compound according to any one of claims 48 to 52, wherein the sugar of the nucleoside at any position downstream of the first nucleoside at the 5'-end region of the first region of the linked nucleosides at the 2nd and 14th positions comprises a 2'-F modification.

54. The oligomeric compound according to any one of claims 52 to 53, wherein, among the nucleosides in the second region of the linked nucleosides, the sugar of the nucleoside at the position corresponding to the nucleoside at any position from the 11th to 13th positions downstream of the first nucleoside at the 5'-end region of the first region of the linked nucleosides comprises a 2'-F modification.

55. The oligomeric compound according to claim 53 or 54, wherein the 3'-terminal nucleoside of the second region of the linked nucleosides comprises a 2'-F modification.

56. The oligomeric compound according to any one of claims 52 to 55, which depends on claim 10, wherein one or more odd-position nucleosides starting from the 5'-terminal region of the first region of the linked nucleosides are modified, and / or one or more even-position nucleosides starting from the 5'-terminal region of the first region of the linked nucleosides are modified, wherein the modification of the even-position nucleosides is generally a second modification different from the modification of the odd-position nucleosides.

57. The oligomeric compound according to claim 56, wherein one or more odd-position nucleosides starting from the 3'-terminal region of the second region of the linked nucleosides are modified in a modification manner different from the modification of the odd-position nucleosides in the first region of the linked nucleosides.

58. The oligomeric compound according to claim 56 or 57, wherein, One or more even-position nucleosides starting from the 3'-terminal region of the second region of the linked nucleosides are modified in a modification manner different from the modification manner of the even-position nucleosides in the first region of the linked nucleosides according to claim 51.

59. The oligomeric compound according to any one of claims 56 to 58, wherein, At least one or more modified even-position nucleosides in the first region of the linked nucleosides are adjacent to at least one or more odd-position nucleosides in the first nucleoside region that are modified in a different modification manner.

60. The oligomeric compound according to any one of claims 56 to 59, wherein, At least one or more modified even-position nucleosides in the second region of the linked nucleosides are adjacent to at least one or more odd-position nucleosides in the second region of the linked nucleosides that are modified in a different modification manner.

61. The oligomeric compound according to any one of claims 56 to 60, wherein, The sugars of one or more odd-position nucleosides starting from the 5'-terminal region of the first region of the nucleosides are sugars modified with 2'-O-methyl.

62. The oligomeric compound according to any one of claims 56 to 61, wherein, One or more even-position nucleosides starting from the 3'-terminal region of the first region of the linked nucleosides are sugars modified with 2'-F.

63. The oligomeric compound according to any one of claims 56 to 62, wherein, The sugars of one or more odd-position nucleosides starting from the 5'-terminal region of the second region of the linked nucleosides are sugars modified with 2'-O-methyl.

64. The oligomeric compound according to any one of claims 56 to 63, wherein, One or more even-position nucleosides starting from the 5'-terminal region of the second region of the linked nucleosides are sugars modified with 2'-F.

65. The oligomeric compound according to any one of claims 45 to 64, wherein, The sugars of multiple adjacent nucleosides in the first nucleoside region are modified in the same or different modification manners.

66. The oligomeric compound according to any one of claims 45 to 65, wherein, The sugars of multiple adjacent nucleosides in the second nucleoside region are modified in the same or different modification manners.

67. The oligomeric compound according to any one of claims 56 to 66, and any one of its dependent claims 30 to 33, wherein, The sugars of multiple adjacent nucleosides in the hairpin loop are modified in the same or different modification manners.

68. The oligomeric compound according to any one of claims 65 to 67, wherein, The same modification is a sugar modified with 2'-F.

69. The oligomeric compound according to any one of claims 65 to 67, wherein, The same modification is a sugar modified with 2'-O-methyl.

70. The oligomeric compound according to claim 69, wherein, The multiple adjacent sugars modified with 2'-O-methyl are present in at least eight adjacent nucleosides in the first and / or second nucleoside regions.

71. The oligomeric compound according to claim 70, wherein, The multiple adjacent sugars modified with 2'-O-methyl are present in three or four adjacent nucleosides in the hairpin loop.

72. The oligomeric compound according to claim 46, any one of its dependent claims 31 to 35, wherein, The hairpin loop comprises at least one nucleoside having a modified sugar.

73. The oligomeric compound according to claim 72, wherein, The at least one nucleoside is adjacent to a nucleoside having a differently modified sugar, and optionally, all adjacent nucleosides in the hairpin loop have differently modified sugars.

74. The oligomeric compound according to claim 73, wherein, The modified sugar is a sugar modified with 2'-O-methyl, and the differently modified sugar is a sugar modified with 2'-F.

75. The oligomeric compound according to any one of claims 1 to 74, wherein, One or more nucleosides in the first region of the linked nucleosides and / or the second region of the linked nucleosides are reverse nucleosides and are linked through the 3'-carbon of their sugar to the 3'-carbon of the sugar of an adjacent nucleoside; and / or one or more nucleosides in the first region of the linked nucleosides and / or the second region of the linked nucleosides are reverse nucleosides and are linked through the 5'-carbon of their sugar to the 5'-carbon of the sugar of an adjacent nucleoside.

76. The oligomeric compound according to any one of claims 1 to 75, having blunt ends.

77. The oligomeric compound according to any one of claims 1 to 76, wherein, The first or second nucleoside region has a protruding end.

78. The oligomeric compound according to any one of claims 1 to 77, wherein, The first region is selected from the group consisting of the sequences of SEQ ID NO: 327, 352, 356, 362, 375, 393 and fragments thereof.

79. The oligomeric compound according to any one of claims 1 to 79, wherein, The second region is selected from the group consisting of the sequences of SEQ ID NO: 427, 452, 456, 462, 475, 493 and fragments thereof, wherein the second region optionally has a length of 14 nucleosides.

80. The oligomeric compound according to any one of claims 1 to 80, wherein, The total length of the oligomeric compound is about 25 to about 37 nucleosides, particularly about 33 or about 34 nucleosides.

81. The oligomeric compound according to any one of claims 10 to 80, wherein, The terminal nucleoside at the 5'-end position of the first region has a nucleobase selected from A, U, G, and C, optionally U; and optionally, the terminal nucleoside at the 3'-end position of the second region is replaced by a base complementary to the base at the 5'-end position of the first region, optionally A.

82. A nucleic acid construct comprising at least: (a) A first nucleic acid portion that is at least partially complementary to at least a first portion of an RNA transcribed from the AGT gene; (b) A second nucleic acid portion that is at least partially complementary to at least a second portion of an RNA transcribed from the AGT gene or a different target gene, the second portion being different from the first portion; (c) A third nucleic acid portion that is at least partially complementary to the first nucleic acid portion in (a) so as to form a first nucleic acid double-stranded region therewith; (d) A fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion in (b) so as to form a second nucleic acid double-stranded region therewith.

83. The construct according to claim 82, wherein the construct is designed such that after in vivo administration, the construct dissociates to produce at least first and second discrete nucleic acid targeting molecules that respectively target the RNA portions transcribed from the target genes in (a) and (b); whereby, (i) the first nucleic acid targeting molecule is capable of regulating the expression of the target gene in (a) and comprises or is derived from at least the first nucleic acid portion in (a); (ii) the second nucleic acid targeting molecule is capable of regulating the expression of the target gene in (b) and comprises or is derived from the second nucleic acid portion in (b).

84. The construct according to claim 82 or 83, wherein the construct is designed to dissociate such that the first and second discrete nucleic acid targeting molecules are respectively processed by independent RNAi-induced silencing complexes.

85. The construct according to any one of claims 82 to 84, further comprising at least one labile functional group such that after in vivo administration, the construct is cleaved to produce the at least first and second discrete nucleic acid targeting molecules.

86. The construct according to claim 85, wherein the labile functional group comprises one or more unmodified nucleotides.

87. The construct according to claim 86, wherein the one or more unmodified nucleotides of the labile functional group represent one or more cleavage sites within the construct, whereby after in vivo administration, the construct is cleaved at the one or more cleavage sites to produce the at least first and second discrete nucleic acid targeting molecules.

88. The construct according to claim 87, wherein the cleavage sites are respectively located within the construct such that after cleavage, the first discrete nucleic acid targeting molecule comprises or is derived from the first nucleic acid duplex region, and the second discrete nucleic acid targeting molecule comprises or is derived from the second nucleic acid duplex region.

89. The construct according to claim 88, wherein the first discrete nucleic acid targeting molecule comprises or consists of the first nucleic acid portion in (a) and the third nucleic acid portion in (c), and / or the second discrete nucleic acid targeting molecule comprises or consists of the second nucleic acid portion in (b) and the fourth nucleic acid portion in (d).

90. The construct according to any one of claims 82 to 89, wherein (a) the nucleobase sequence of the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1 to 100; (b) the nucleobase sequence of the second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1 to 100; (c) the nucleobase sequence of the third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 101 to 200; and / or (d) the nucleobase sequence of the fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 101 to 200. Wherein the third and fourth nucleobase sequences may be one, two or three nucleobases less in terms of having a length of 14 nucleobases, wherein optionally the nucleobase at the 5′ end is missing.

91. The construct according to any one of claims 82 to 90, wherein the first nucleic acid portion in (a) is directly or indirectly linked in primary structure to the fourth nucleic acid portion in (d).

92. The construct according to claim 91, wherein the nucleobase sequences of the first and fourth nucleic acid moieties are selected from the group consisting of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141, 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and 200, 40 and 140, 75 and 175, 30 and 130, 42 and 142, 81 and 181, 17 and 117, 34 and 134, 53 and 153, 29 and 129, 26 and 126, 74 and 174, 94 and 194, 14 and 114, 3 and 103, 7 and 107, and 2 and 102; wherein, Optionally, the sequences of SEQ ID NOs: 27, 52, 56, 62, 75 and 93 may be one, two, three or four nucleobases less, and optionally the nucleobase at the 5′ end is missing.

93. The construct according to any one of claims 82 to 92, wherein the second nucleic acid portion in (b) is directly or indirectly linked in primary structure to the third nucleic acid portion in (c).

94. The construct according to claim 92 or 93, wherein the nucleobase sequences of the second and third nucleic acid portions are selected from the group consisting of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141, 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and 200, 40 and 140, 75 and 175, 30 and 130, 42 and 142, 81 and 181, 17 and 117, 34 and 134, 53 and 153, 29 and 129, 26 and 126, 74 and 174, 94 and 194, 14 and 114, 3 and 103, 7 and 107, and 2 and 102; wherein, Optionally, the sequences of SEQ ID NOs: 27, 52, 56, 62, 75 and 93 may be one, two, three or four nucleobases less, and optionally the nucleobase at the 5′ end is missing.

95. The construct according to any one of claims 82 to 90, 91 or 93, further comprising 1 to 8 additional nucleic acid portions, each of which is at least partially complementary to 1 to 8 additional portions of RNA transcribed from one or more target genes, which target genes may be the same or different from each other and / or the same or different from the target genes defined in (a) and / or (b); and wherein each of the 1 to 8 additional nucleic acid portions forms an additional duplex region with a respective transient nucleic acid portion that is at least partially complementary thereto.

96. The construct according to claim 95, wherein the second nucleic acid moiety in (b) and the 1 to 8 additional nucleic acid moieties are directly or indirectly linked to a selected passenger nucleic acid moiety in their respective primary structures.

97. The construct according to any one of claims 91, 93 or 96, wherein said direct or indirect linkage represents (i) an internucleotide bond, (ii) an internucleotide gap, or (iii) a nucleic acid linker moiety consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, said nucleic acid linker moiety being optionally single-stranded.

98. The construct according to claim 97(i), wherein the linkage is direct so as to form a continuous strand.

99. The construct according to any one of claims 82 to 99, in particular the construct according to claim 97(i), wherein there is a certain complementarity between the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b), or between the third nucleic acid moiety in (c) and the fourth nucleic acid moiety in (d).

100. The construct according to claim 99, wherein the complementarity: (i) is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or 2, 3, 4 or 5 base pairs; and / or (ii) exists between the first nucleic acid moiety in (a) and the second nucleic acid moiety in (b).

101. The construct according to claims 97(i) to 100, which are dependent on claim 86, wherein the internucleotide bond involves at least one of said one or more unmodified nucleotides, wherein optionally cleavage occurs at the 3'-position of (at least one) said unmodified nucleotide.

102. The construct according to any one of claims 82 to 101, wherein the length of the first nucleic acid moiety in (a), and / or the second nucleic acid moiety in (b), and / or the third nucleic acid moiety in (c), and / or the fourth nucleic acid moiety in (d) is 7 to 25 nucleotides, respectively.

103. The construct according to claim 102, wherein the length of the first nucleic acid moiety in (a) and / or the second nucleic acid moiety in (b) is 18 to 21, 18 to 20 or 19 nucleotides.

104. The construct according to claim 102 or 103, wherein the length of the third nucleic acid moiety in (c) and / or the fourth nucleic acid moiety in (d) is 11 to 20, 13 to 16, 14 or 15, or 14 nucleotides.

105. The construct according to any one of claims 102 to 104, wherein the unmodified nucleotide is located at any position in the 18 to 25 positions, any position in the 18 to 21 positions, and / or the 3'-terminal position of the first nucleic acid moiety in (a) and / or the third nucleic acid moiety in (c).

106. The construct according to claim 105, wherein the unmodified nucleotide is located at the 19th position.

107. A construct according to any one of claims 99 to 100 or 102 to 105, which are dependent on claim 87(iii), wherein the nucleic acid linker portion has a length of 1 to 8 nucleotides, 2 to 7 nucleotides, 3 to 6 nucleotides, or 4 or 5 nucleotides.

108. A construct according to any one of claims 103 to 107, wherein one or more of all the double-stranded regions each independently have a length of 10 to 19 base pairs, 13 to 19 base pairs, 13 base pairs, 14 or 15 base pairs, or 14 base pairs, and optionally, there is one mismatch within the double-stranded region.

109. A construct according to any one of claims 82 to 108, further comprising one or more ligands.

110. A construct according to any one of claims 82 to 109, wherein the first nucleic acid portion in (a), and / or the second nucleic acid portion in (b), and / or the third nucleic acid portion in (c), and / or the fourth nucleic acid portion in (d), and, if present, the 1 to 8 additional nucleic acid portions as defined in claims 95 and 96, and / or the passenger nucleic acid portion each have a 5′ to 3′ directionality, thereby defining their 5′ region and 3′ region.

111. A construct according to any one of claims 109 or 110, wherein one or more ligands are conjugated at the 3′ region (optionally the 3′ end) of any of the following portions: (i) the third nucleic acid portion in (c); and / or (ii) the fourth nucleic acid portion in (d); and, if present, (iii) the passenger nucleic acid portion as defined in claim 95 or 96.

112. A construct according to any one of claims 109 to 111, wherein one or more ligands are conjugated at one or more regions between the 5′ region and the 3′ region of any of the nucleic acid portions (optionally the third nucleic acid portion in (c), and / or the fourth nucleic acid portion in (d), and / or the passenger nucleic acid portion as defined in claim 95 or 96).

113. A construct according to any one of claims 109 to 112, wherein one or more ligands are conjugated at the 5′ region (optionally the 5′ end) of any of the nucleic acid portions.

114. A construct according to any one of claims 109 to 113, wherein the one or more ligands are any cell-targeting moieties, such as lipids, carbohydrates, aptamers, vitamins, and / or peptides that bind to specific targets on the cell membrane or cell surface.

115. A construct according to claim 114, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

116. A construct according to claim 115, wherein the one or more carbohydrates comprise one or more hexose moieties.

117. A construct according to claim 116, wherein the one or more hexose moieties are one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

118. The construct according to claim 117, comprising two or three N - acetylgalactosamine moieties.

119. The construct according to any one of claims 109 to 118, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

120. The construct according to claim 119, wherein the one or more ligands are linked in a bi - antenna or tri - antenna configuration, or in a configuration based on individual ligands at different positions.

121. The construct according to claim 118 or 119, wherein the ligand has the following structure:

122. The construct according to any one of claims 82 to 121, further comprising one or more phosphorothioate or phosphorodithioate internucleotide linkages.

123. The construct according to claim 122, comprising 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.

124. The construct according to claim 122 or 123, comprising one or more phosphorothioate or phosphorodithioate internucleotide linkages in one or more 5' regions and / or 3' regions of the following parts: the first nucleic acid part in (a); and / or the second nucleic acid part in (b); and / or the third nucleic acid part in (c); and / or the fourth nucleic acid part in (d); and / or the 1 to 8 additional nucleic acid parts as defined in claim 95 or 96; and / or the passenger nucleic acid part as defined in claim 95 or 96.

125. The construct according to any one of claims 122 to 124, comprising phosphorothioate or phosphorodithioate internucleotide linkages between at least two adjacent nucleotides of the nucleic acid linking part as defined in claim 97(iii).

126. The construct according to claim 125, comprising phosphorothioate or phosphorodithioate internucleotide linkages between each adjacent nucleotide present in the nucleic acid linking part.

127. The construct according to any one of claims 122 to 126, comprising phosphorothioate or phosphorodithioate internucleotide linkages for linking: (a) the first nucleic acid part to the nucleic acid linking part as defined in claim 97(iii); and / or (b) the second nucleic acid part to the nucleic acid linking part as defined in claim 97(iii); and / or (c) the third nucleic acid part to the nucleic acid linking part as defined in claim 97(iii); and / or (d) the fourth nucleic acid part to the nucleic acid linking part as defined in claim 97(iii); and / or the 1 to 8 additional nucleic acid parts as defined in claim 95 or 96 to the nucleic acid linking part as defined in claim 97(iii); and / or the passenger nucleic acid part as defined in claim 95 or 96 to the nucleic acid linking part as defined in claim 97(iii).

128. The construct according to any one of claims 82 to 127, wherein at least one nucleotide in at least one of the following parts is modified: (a) the first nucleic acid part; and / or (b) the second nucleic acid part; and / or the third nucleic acid portion in (c); and / or the fourth nucleic acid portion in (d); and / or if present, 1 to 8 additional nucleic acid portions as defined in claim 95 or 96; and / or if present, the passenger nucleic acid portion as defined in claim 95 or 96; and / or if present, the nucleic acid linking portion as defined in claim 97(iii).

129. The construct according to claim 128, wherein one or more odd-position nucleotides starting from the 5'-region of one of the following portions are modified, and / or one or more even-position nucleotides starting from the 5'-region of one of the following portions are modified, wherein the modification of the even-position nucleotides is generally a second modification different from the modification of the odd-position nucleotides: (a) the first nucleic acid portion; and / or (b) the second nucleic acid portion; and / or (c) the third nucleic acid portion; and / or (d) the fourth nucleic acid portion; and / or if present, 1 to 8 additional nucleic acid portions as defined in claim 95 or 96; and / or if present, the passenger nucleic acid portion as defined in claim 95 or 96.

130. The construct according to claim 128 or 129: one or more odd-position nucleotides starting from the 3'-region of the third nucleic acid portion in (c) are modified in a manner different from the modification of the odd-position nucleotides starting from the 5'-region of the first nucleic acid portion in (a); and / or one or more odd-position nucleotides starting from the 3'-region of the fourth nucleic acid portion in (d) are modified in a manner different from the modification of the odd-position nucleotides starting from the 5'-region of the second nucleic acid portion in (b); and / or if present, one or more odd-position nucleotides starting from the 3'-region of the passenger nucleic acid portion as defined in claim 95 or 96 are modified in a manner different from the modification of the odd-position nucleotides starting from the 5'-region of the 1 to 8 additional nucleic acid portions as defined in claim 95 or 96; and / or if present, one or more nucleotides of the nucleic acid linking portion as defined in claim 97(iii) are modified by the following modification methods: (i) different from the modification of the adjacent nucleotide in the 3'-region of the first nucleic acid portion in (a); and / or (ii) different from the modification of the adjacent nucleotide in the 3'-region of the second nucleic acid portion in (b); and / or different from the modification of the adjacent nucleotide in the 3'-region of the 1 to 8 additional nucleic acid portions as defined in claim 95 or 96 (if present).

131. The construct according to any one of claims 128 to 130, wherein one or more even-position nucleotides starting from the 3'-region of the following portions are modified in a manner different from the modification of the odd-position nucleotides starting from the 3'-region of their respective portions: (i) the third nucleic acid portion in (c); and / or (ii) the fourth nucleic acid portion in (d); and / or (iii) if present, the passenger nucleic acid portion as defined in claim 95 or 96.

132. The construct according to any one of claims 128 to 131, wherein at least one or more of the following parts have modified even-position nucleotides adjacent to at least one or more of the odd-position nucleotides modified in a different manner in these respective parts: (i) the first nucleic acid part in (a); and / or (ii) the second nucleic acid part in (b); and / or (iii) if present, 1 to 8 additional nucleic acid parts as defined in claim 95 or 96.

133. The construct according to any one of claims 128 to 132, wherein at least one or more of the following parts have modified even-position nucleotides adjacent to at least one or more of the odd-position nucleotides modified in a different manner in these respective parts: (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the passenger nucleic acid part as defined in claim 95 or 96.

134. The construct according to any one of claims 128 to 133, wherein a plurality of adjacent nucleotides in the following parts are modified by the same modification method: (i) the first nucleic acid part in (a); and / or (ii) the second nucleic acid part in (b); and / or (iii) if present, 1 to 8 additional nucleic acid parts as defined in claim 95 or 96.

135. The construct according to any one of claims 128 to 134, wherein a plurality of adjacent nucleotides in the following parts are modified by the same modification method: (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the passenger nucleic acid part as defined in claim 95 or 96.

136. The construct according to claim 134 or 135, wherein the plurality of adjacent nucleotides modified in the same manner are 2 to 4 adjacent nucleotides, or 3 or 4 adjacent nucleotides.

137. The construct according to claim 136, wherein the plurality of adjacent nucleotides modified in the same manner are located in the 5'-region of the following parts: (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the passenger nucleic acid part as defined in claim 95 or 96.

138. The construct according to any one of claims 134 to 137, wherein the plurality of adjacent nucleotides modified in the same manner are located in the nucleic acid linking part as defined in claim 97(iii).

139. The construct according to any one of claims 128 to 138: (a) One or more modified nucleotides of the first nucleic acid part do not have the same modification as the corresponding nucleotides of the third nucleic acid part in (c) in the first double-stranded region; and / or (b) One or more modified nucleotides of the second nucleic acid part do not have the same modification as the corresponding nucleotides of the fourth nucleic acid part in (d) in the second double-stranded region; and / or If present, one or more modified nucleotides of the 1 to 8 additional nucleic acid moieties defined in claim 95 or 96 do not have the same modification as the corresponding nucleotide of the corresponding passenger nucleic acid moiety in the respective double-stranded region.

140. The construct according to any one of claims 128 to 139: (a) one or more modified nucleotides of the first nucleic acid moiety are offset by at least one nucleotide relative to a common modified nucleotide of the third nucleic acid moiety in (c); and / or (b) one or more modified nucleotides of the second nucleic acid moiety are offset by at least one nucleotide relative to a common modified nucleotide of the fourth nucleic acid moiety in (d); and / or If present, one or more modified nucleotides of the 1 to 8 additional nucleic acid moieties defined in claim 95 or 96 are offset by at least one nucleotide relative to a common modified nucleotide of the (if present) passenger nucleic acid moiety defined in claim 95 or 96.

141. The construct according to any one of claims 128 to 140, wherein the one or more modifications are each independently a sugar modification, a phosphate modification or a base modification.

142. The construct according to claim 141, wherein the modification is selected from nucleotides having a 2'-modified sugar; conformationally restricted nucleotide (CRN) sugars such as locked nucleic acid (LNA), (S)-constrained ethyl bicyclic nucleic acid, constrained ethyl (cEt), tricyclic DNA; morpholino, unlocked nucleic acid (UNA), glycerol nucleic acid (GNA), D-hexitol nucleic acid (HNA) and cyclohexene nucleic acid (CeNA).

143. The construct according to claim 142, wherein the 2′-modified sugar is selected from 2'-O-alkyl-modified sugars, 2′-O-methyl-modified sugars, 2′-O-methoxyethyl-modified sugars, 2'-O-allyl-modified sugars, 2′-C-allyl-modified sugars, 2′-deoxy-modified sugars (such as 2'-deoxyribose), 2′-F-modified sugars, 2′-arabino-fluoro-modified sugars, 2'-O-benzyl-modified sugars, 2'-amino-modified sugars and 2'-O-methyl-4-pyridyl-modified sugars.

144. The construct according to any one of claims 141 to 143, wherein the base modification is either a abasic nucleotide or a nucleotide comprising a non-natural base.

145. The construct according to any one of claims 138 to 144, wherein at least one modification is a 2′-O-methyl modification of the ribose moiety.

146. The construct according to any one of claims 138 to 145, wherein at least one modification is a 2′-F modification of the ribose moiety.

147. The construct according to any one of claims 138 to 146, wherein the nucleotide at any position among the 2nd and 14th positions downstream of the first nucleotide in the 5' region of the following moieties does not contain a 2'-O-methyl modification in its ribose moiety: (i) the first nucleic acid moiety in (a); and / or (ii) the second nucleic acid moiety in (b); and / or (iii) if present, the 1 to 8 additional nucleic acid moieties defined in claim 95 or 96.

148. The construct according to any one of claims 138 to 147, wherein one, two or all three nucleotides at positions corresponding to the nucleotides at any position from the 11th to the 13th positions downstream of the first nucleotide in the 5'-region of the following part respectively, have ribose moieties that do not contain a 2'-O-methyl modification: (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the pass-through nucleic acid part as defined in claim 95 or 96; wherein the corresponding part is (i) the first nucleic acid part in (a); and / or (ii) the second nucleic acid part in (b); and / or (iii) if present, 1 to 8 additional nucleic acid parts as defined in claim 95 or 96.

149. The construct according to claim 147 or 148, wherein the nucleotide at any position from the 2nd to the 14th positions downstream of the first nucleotide in the following part has a ribose moiety that contains a 2'-F modification: (i) the first nucleic acid part in (a); and / or (ii) the second nucleic acid part in (b); and / or (iii) if present, 1 to 8 additional nucleic acid parts as defined in claim 95 or 96.

150. The construct according to any one of claims 147 to 149, wherein one, two or all three nucleotides at positions corresponding to the nucleotides at any position from the 11th to the 13th positions downstream of the first nucleotide in the 5'-region of the following part respectively, have ribose moieties that contain a 2'-F modification: (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the pass-through nucleic acid part as defined in claim 95 or 96; wherein the corresponding part is (i) the first nucleic acid part in (a); and / or (ii) the second nucleic acid part in (b); and / or (iii) if present, 1 to 8 additional nucleic acid parts as defined in claim 95 or 96.

151. The construct according to any one of claims 146 to 150, wherein, except for the unmodified nucleotides according to claim 86, the ribose moieties of all the remaining nucleotides contain a 2'-O-methyl modification or a 2'-F modification.

152. The construct according to claim 151, wherein the ribose moieties of the remaining nucleotides contain a 2'-O-methyl modification.

153. The construct according to claim 151 or 152, wherein the one or more (optionally one) unmodified nucleotides represent any nucleotide of the nucleic acid linking part as defined in claim 97(iii), optionally a nucleotide adjacent to the following part in the nucleic acid linking part as defined in claim 97(iii): (i) the third nucleic acid part in (c); and / or (ii) the fourth nucleic acid part in (d); and / or (iii) if present, the pass-through nucleic acid part as defined in claim 95 or 96.

154. The construct according to any one of the preceding claims, wherein (a) The first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 501 - 600 and 3604 - 3605; (b) The second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 501 - 600 and 3604 - 3605; (c) The third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401 - 500; and / or (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401 - 500.

155. The construct according to claim 154, wherein the 3' - terminal positions of the first and third nucleic acid portions are substituted with unmodified nucleotides.

156. The construct according to any one of claims 82 to 155, comprising at least one vinyl phosphonate modification, for example, comprising at least one vinyl phosphonate modification in the 5′ - region of the following portions: (i) the first nucleic acid portion in (a); and / or (ii) the second nucleic acid portion in (b); and / or (iii) if present, 1 to 8 additional nucleic acid portions as defined in claims 95 or 96.

157. The construct according to any one of claims 82 to 156, wherein one or more nucleotides in the following portions are inverted nucleotides, and the inverted nucleotide is linked to the 3′ - carbon of an adjacent nucleotide through its 3' - carbon, and / or the inverted nucleotide is linked to the 5′ - carbon of an adjacent nucleotide through its 5' - carbon: the first nucleic acid portion in (a); and / or the second nucleic acid portion in (b); and / or the third nucleic acid portion in (c); and / or the fourth nucleic acid portion in (d); and / or if present, 1 to 8 additional nucleic acid portions as defined in claims 95 or 96; and / or if present, the passenger nucleic acid portion as defined in claims 95 or 96.

158. The construct according to claim 157, wherein the inverted nucleotide is linked to an adjacent nucleotide through a phosphodiester bond via a phosphate group; or through a phosphorothioate group to an adjacent nucleotide; or through a phosphorodithioate group to an adjacent nucleotide.

159. The construct according to any one of claims 82 to 158, which has a blunt - end structure.

160. The construct according to any one of claims 82 to 158, wherein the following portions have overhanging ends: the first nucleic acid portion in (a); and / or the second nucleic acid portion in (b); and / or the third nucleic acid portion in (c); and / or the fourth nucleic acid portion in (d); and / or if present, 1 to 8 additional nucleic acid portions as defined in claims 95 or 96; and / or if present, the passenger nucleic acid portion as defined in claims 95 or 96.

161. The construct according to any one of claims 82 to 160, wherein the target RNA is messenger RNA (mRNA) or other RNA molecules.

162. The construct according to any one of claims 82 to 160, wherein the second nucleic acid portion in (b) is at least partially complementary to at least a second portion of RNA transcribed from a target gene different from the AGT gene.

163. The construct according to any one of claims 82 to 162, wherein the target gene different from the AGT gene is selected from the following group: - Genes encoding atherogenic lipoproteins containing apolipoprotein B (ApoB); - Genes encoding proteins related to the inflammatory signaling pathway (such as interleukin-6 (IL-6), C-reactive protein (CRP), and - interleukin-11 (IL-11)); - Genes encoding other proteins related to hypertension; - Genes encoding proteins related to platelet aggregation and the coagulation pathway; - Genes encoding proteins related to diabetes; - Genes encoding proteins related to obesity and metabolic syndrome; - Genes encoding proteins related to other atherogenic factors and modifiable risk factors for cardiovascular disease.

164. The construct according to claim 82 or 163, wherein the target gene different from the AGT gene is selected from the following group: - Apolipoprotein C3 (APOC3) gene; - Proprotein convertase subtilisin / kexin type 9 (PCSK9) gene; - Angiopoietin-like protein 3 (ANGPTL3) gene; - Angiopoietin-like protein 4 (ANGPTL4) gene; - Lipoprotein(a) [Lp(a)] gene; - Angiopoietin-like protein 8 (ANGPTL 8) gene; - Asialoglycoprotein receptor 1 / 2 (ASGR1 / 2) gene.

165. The construct according to claim 164, wherein the target gene different from the AGT gene is the APOC3 gene, wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1-100; (b) the second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 601-976; (c) the third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 101-200; (d) the fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1353-1728.

166. The construct according to claim 164 or 165, wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 301-400; (b) the second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 977-1352; (c) the third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401-500; (d) the fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1729-2104.

167. The construct according to any one of claims 164 to 166, wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 1-100; (b) the second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2105-2119; (c) the third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 101-200; (d) the fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2135-2149.

168. The construct according to any one of claims 164 to 167, wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 301-400; (b) the second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2120-2134; (c) the third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401-500; (d) the fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2150-2164.

169. The construct according to any one of claims 164 to 168, wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 301-400; (b) The second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 3450 - 3456; (c) The third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401 - 500; (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 3464 - 3470.

170. The construct according to claim 164, wherein the target gene different from the AGT gene is the PCSK9 gene, and wherein (a) the first nucleic acid portion is selected from the group consisting of SEQ ID NOs: 301 - 400; (b) The second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2189 - 2215; (c) The third nucleic acid portion is selected from the group consisting of SEQ ID NOs: 401 - 500; (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2216 - 2242.

171. The construct according to claim 164 or 170, wherein (a) The first nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 1 - 100 and 301 - 400; (b) The second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2493 - 2742; (c) The third nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 101 - 200 and 401 - 500; (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 2993 - 3242.

172. The construct according to claim 164, 170 or 171, wherein (a) The first nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 1 - 100 and 301 - 400; (b) The second nucleic acid portion is selected from the group consisting of SEQ ID NOs: 3243 - 3328; (c) The third nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 101 - 200 and 401 - 500; (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 3329 - 3414.

173. The construct according to claim 164 or any one of claims 170 to 172, wherein (a) The first nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 1 - 100 and 301 - 400; (b) The second nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 341 - 3421 and 3429 - 3435; (c) The third nucleic acid portion is selected from the groups consisting of SEQ ID NOs: 101 - 200 and 401 - 500; (d) The fourth nucleic acid portion is selected from the group consisting of SEQ ID NOs: 3443 - 3456.

174. The construct according to claims 82 to 173, further comprising 1 to 8 additional nucleic acid portions, each of which is at least partially complementary to 1 to 8 additional portions of the RNA transcribed from one or more target genes, which target genes are different from each other and / or the same as or different from the target genes defined in (a) and (b); and wherein each of the 1 to 8 additional nucleic acid portions forms an additional double-stranded region with a respective passenger nucleic acid portion that is at least partially complementary thereto.

175. The construct according to claim 174, further comprising 1 additional nucleic acid portion.

176. The construct according to claim 175, which targets a target gene selected from the following group: (a) The AGT gene, (b) The APOC3 gene, and (e) The PCSK9 gene; (a) The AGT gene, (b) The ANGPTL3 gene, and (e) The Lp(a) gene; (a) AGT gene, (b) APOC3 gene, and (e) Lp(a) gene; Among them, (a) is a first nucleic acid portion that is at least partially complementary to at least a first portion of RNA transcribed from the AGT gene; (b) is a second nucleic acid portion that is at least partially complementary to a first portion of RNA transcribed from a second gene; (e) is a fifth nucleic acid portion that is at least partially complementary to a third portion of RNA transcribed from a third gene; (f) is a sixth nucleic acid portion that is at least partially complementary to (e); optionally, the target gene is (a) AGT gene, (b) APOC3 gene, and (e) PCSK9 gene.

177. A composition comprising an oligomeric compound according to any one of claims 1 to 81 and / or a nucleic acid construct according to any one of claims 82 to 176, and a physiologically acceptable excipient.

178. A pharmaceutical composition comprising an oligomeric compound according to any one of claims 1 to 81 and / or a nucleic acid construct according to any one of claims 82 to 176.

179. The pharmaceutical composition according to claim 178, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant, and / or preservative.

180. The pharmaceutical composition according to claim 178 or 179, wherein the oligomeric compound according to any one of claims 1 to 81 and / or the construct according to any one of claims 82 to 176 is the sole pharmaceutically active ingredient.

181. The pharmaceutical composition according to claim 178 or 179, wherein the pharmaceutical composition further comprises one or more other pharmaceutically active ingredients.

182. The pharmaceutical composition according to claim 181, wherein the other pharmaceutically active ingredient is a drug for reducing hypertension, and the other pharmaceutically active ingredient is optionally selected from diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, β-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists, non-selective α-adrenergic antagonists, synthetic steroidal mineralocorticoid drugs; combinations of any of the above drugs; and antihypertensive drugs formulated as a combination preparation; more optionally, the angiotensin II receptor antagonist is selected from losartan, valsartan, olmesartan, eprosartan, and azilsartan.

183. The pharmaceutical composition according to claim 181 or 182, wherein the other pharmaceutically active ingredient is another oligomeric compound targeting a target different from APOC3, optionally targeting PCSK9; ethyl eicosapentaenoate (Vascepa); vupanorsen; statins such as rosuvastatin and simvastatin; fibrates such as fenofibrate; and / or a compound for reducing low-density lipoprotein cholesterol (LDL-cholesterol), such as statins and ezetimibe.

184. The pharmaceutical composition according to any one of claims 181 or 182, wherein the other pharmaceutically active ingredient is another oligomeric compound targeting a target different from PCSK9, and / or a lipid-lowering agent different from the oligomeric compound, wherein the lipid-lowering agent is optionally a statin or ezetimibe.

185. The pharmaceutical composition according to claims 181 to 184, wherein the oligomeric compound and / or the nucleic acid construct, and the other pharmaceutically active ingredient can be administered simultaneously, or in any order.

186. The oligomeric compound according to any one of claims 1 to 81 and / or the nucleic acid construct according to any one of claims 82 to 176, for use in human or veterinary medicine or therapy.

187. The oligomeric compound according to any one of claims 1 to 81 and / or the nucleic acid construct according to any one of claims 82 to 176, for use in a method of treating, ameliorating, and / or preventing a disease or disorder.

188. The compound and / or construct for use in treatment according to claim 187, wherein the disease or disorder is a disease or disorder associated with AGT, or a disease or disorder that requires reduction of AGT expression.

189. The compound and / or construct for use in treatment according to claim 188, wherein the disease or disorder is selected from the group consisting of: hypertension, hypertensive disease, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, elevated intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypotension, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth retardation, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes, and metabolic syndrome.

190. A compound and / or construct for treatment according to claim 188 or 189, wherein the disease or disorder is also a disease or disorder associated with APOC3, or a disease or disorder that requires reduction of APOC3 expression level, and the disease or disorder is optionally selected from dyslipidemia (including mixed dyslipidemia); hyperchylomicronemia (including familial hyperchylomicronemia); hypertriglyceridemia (optionally severe hypertriglyceridemia and / or hypertriglyceridemia with blood triglyceride level higher than 500 mg / dl); inflammation (including mild inflammation); atherosclerosis; atherosclerotic cardiovascular disease (ASCVD), including major adverse cardiovascular events (MACE), such as myocardial infarction, stroke and peripheral arterial disease; and pancreatitis (including acute pancreatitis).

191. A compound and / or construct for treatment according to claims 188 to 190, wherein the disease or disorder is also a disease or disorder associated with PCSK9, or a disease or disorder that requires reduction of low-density lipoprotein (LDL) cholesterol, and the disease or disorder is optionally selected from dyslipidemia (including mixed dyslipidemia), hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atherosclerotic cardiovascular disease (ASCVD), including myocardial infarction, stroke and peripheral arterial disease.

192. A method for treating a disease or disorder, comprising administering to an individual in need of treatment an oligomeric compound according to any one of claims 1 to 81 and / or a nucleic acid construct according to any one of claims 82 to 176.

193. The method according to claim 192, wherein the oligomeric compound and / or the nucleic acid construct is administered to the individual by subcutaneous or intravenous route.

194. Use of an oligomeric compound according to any one of claims 1 to 81 or a nucleic acid construct according to any one of claims 82 to 176 as a gene function analysis tool in research.

195. Use of an oligomeric compound according to any one of claims 1 to 81 and / or a nucleic acid construct according to any one of claims 82 to 176 in the preparation of a medicament for treating a disease or disorder.

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